Peptide having anti-thrombus activity and method of producing the same

ABSTRACT

A multimer peptide from a snake venom has an activity to inhibit binding between von Willebrand factor and platelets. The multimer peptide is used to obtain a single strand peptide which does not substantially cause decrease in platelets at a minimum dose for exhibiting the activity in vivo. The single strand peptide is obtained by allowing the multimer peptide to exist together with a protein-denaturing agent, and glutathione and/or cysteine, and thereby disconnecting disulfide bonds between peptide chains for constituting the multimer peptide while substantially preserving disulfide bonds within the peptide chains. Alternatively, the single strand peptide, a mutant thereof, or a part thereof is produced by genetic engineering techniques by using genes coding for them.

TECHNICAL FIELD

The present invention relates to a peptide having anti-thrombusactivity, a method of producing the peptide, and a pharmaceuticalcomposition containing the peptide. In particular, the present inventionrelates to a peptide originating from a snake venom, the peptide notcausing decrease in platelets or thrombocytes (thrombocytopenia) invivo.

BACKGROUND ART

It is widely known that platelets closely participate in crisis ofso-called thrombosis represented by myocardial infarction and cerebralthrombosis ("Platelets", edited by Yamanaka and Yamazaki, Igaku-Syoin,pp. 158-163 (1991)). Recently, it has been reported that the bindingbetween von Willebrand factor as one of blood proteins and glycoproteinIb located on platelet surfaces is important for platelets to adhere tointravascular subendotherial tissue, which is considered as an earlyreaction to cause thrombosis (J. P. Cean et al., J. Lab. Clin. Med., 87,586-596 (1976)).

It is known that the binding between the two species of the proteinsdoes not occur in an ordinary state, but it occurs only when a highshear stress is exerted in vivo (T. T. Vincent et al., Blood, 65,823-831 (1985)). The methodology to observe the binding exo-vivoincludes a widely spread method which uses certain substances such asristocetin as an antibiotic (M. A. Howard, B. G. Firkin, Thromb.Haemostatis, 26, 362-369 (1971)) and botrocetin as a protein originatingfrom a snake venom (M. S. Read et al., Proc. Natl. Acad. Sci. U.S.A.,75, 4514-4518 (1978)). Platelet aggregation occurs when these substancesare added to a suspension of platelets. These aggregation depends on thebinding between von Willebrand factor and glycoprotein Ib (M. A. Howard,B. G. Firkin, M. S. Read et al., supra).

Several compounds have been already reported, which exhibit aninhibiting action on the platelet aggregation mediated by ristocetin orbotrocetin. Such known compounds include, for example,aurintricarboxylic acid (M. D. Phillips et al., Blood, 72, 1989-1903(1988)), and dye substances such as aromatic amidino compounds (J. D.Geratz et al., Thromb. Haemostasis, 39, 411-425 (1978)), as well aspartial fragment peptides of von Willebrand factor or glycoprotein Ib(Y. Fujimura et al., J. Biol. Chem., 261, 381-385 (1986); K. Titani etal., Proc. Natl. Acad. Sci. U.S.A., 84, 5610-5614 (1987)).

It has been also reported that peptides having similar plateletaggregation-inhibiting activities are present in snake venoms. Aninternational publication pamphlet of WO9208472 describes such peptidesfrom Crotalus horridus horridus and Cerastes cerastes, each peptidecomprising two different strands having a molecular weight of about 25kilodaltons in which the homology is high at least in amino acidsequences on N-terminal side. A platelet aggregation-inhibiting peptide,which has been reported by Peng et al. (M. Peng et al., Blood, 81,2321-2328 (1993)) as obtained from Echis carinatus, is also extremelysimilar to the peptides described above in its in vitro activity,molecular weight, etc. Any of the platelet aggregation-inhibitingpeptides originating from snake venoms inhibits platelet aggregationmediated by ristocetin or botrocetin at a low concentration of 2 to 5μg/ml or less in vitro.

The international publication pamphlet of WO9208472 does not describesthe anti-thrombus activity upon administration to animals. Accordingly,as described in Example 1 in this specification, the present inventorspurified a peptide having equivalent properties to the peptide describedin the pamphlet, considering a purification method for the peptideoriginating from Crotalus horridus horridus described in the pamphlet,in order to investigate the action upon administration to animals. As aresult, the present inventors observed almost complete disappearance ofplatelets in blood upon administration in a small amount of 100 μg/kg.However, the international publication pamphlet of WO9208472 describesneither suggestion nor solution of such a problem upon administration toanimals.

Moreover, the peptide from Echis carinatus obtained by Peng et al. hasits molecular weight of about 26 kilodaltons under a non-reducedcondition as measured by SDS-polyacrylamide gel electrophoresis, whilethe peptide provides two peptides of about 14 kilodaltons and about 16kilodaltons under a reduced condition. Accordingly, it is postulatedthat this peptide is also homologous to the peptide originating fromCrotalus horridus horridus. It has been also reported for this peptidethat remarkable decrease in platelets is observed upon administration toanimals (M. Peng et al., Blood, 81, 2321-2328 (1993)).

The respective peptides originating from snake venoms, which inhibit thebinding between von Willebrand factor and platelets, have high homologyin their amino acid sequences, and they are also extremely similar intheir molecular weights. In addition, according to the result describedabove, it is assumed that these peptides also have an activity to causedecrease in platelets in vivo. Namely, these peptides inhibit thebinding of von Willebrand factor with platelets in vitro at a lowconcentration, however, they are difficult to be utilized as ananti-thrombosis drug for in vivo administration. The present inventorshave considered the mechanism to cause the decrease in platelets asfollows.

The peptides from snake venoms, which inhibit platelet aggregationmediated by ristocetin or botrocetin at a low concentration, have, forexample, an amino acid sequence as described in the internationalpublication pamphlet of WO9208472. According to the amino acid sequence,the peptide from snake venom conserves approximate positions of cysteineresidues and a part of a sequence considered to be necessary for thelectin activity (W. I. Weis et al., Science, 254, 1608-1615 (1991)), ascompared with a peptide having a calcium-dependent lectin activityreported by Drickamer et al. and referred to as "C-type lectin" (K.Drickamer, J. Biol. Chem., 263, 9557-9560 (1988)).

Other than the above, peptides having high homology to the C-type lectinin their amino acid sequences have been obtained from snake venoms,including, for example, botrocetin obtained from Botrops jararaca (Y.Usami et al., Proc. Natl. Acad. Sci. U.S.A., 90, 928-932 (1993)), rattlesnake lectin obtained from Crotalus atrox (J. Hirabayashi et al., J.Biol. Chem., 266, 2320-2326 (1991)), and alboaggregin obtained fromTrimeresurus albolabris (E. Yoshida et al., Biochem. Biophys. Res.Commun., 191, 1386-1392 (1993)).

These peptides also conserve approximate positions of cysteine residuesand a part of the sequence considered to be necessary for the lectinactivity, as compared with C-type lectin. It is also known that C-typelectin has an activity to agglutinate cells, bacteria and so on throughglycoproteins (sugar chains) on their cell membranes (K. Drickamer, J.Hirabayashi et al., supra).

When the platelet-aggregation is measured in vitro, collected bloodsamples are often added with citric acid, sodium citrate, andethylenediaminetetraacetic acid (EDTA) as anticoagulants, and hencecalcium is chelated in such a platelet aggregation-measuring system.Accordingly, calcium-dependent reactions are inhibited. For this reason,it is difficult to detect in vitro whether or not the peptides fromsnake venoms exhibiting the platelet aggregation-inhibiting activityhave the lectin activity.

Thus it is considered that the peptides from snake venoms express theC-type lectin activity in the presence of calcium in vivo uponadministration to animals, and they cause aggregation of platelets. Itis assumed that such an action is one of factors to cause the observedphenomena such as collection of platelet aggregates in microvessels, andconsequent decrease in platelets or thrombocytes (thrombocytopenia).

Therefore, in order that the peptide from snake venom which inhibits thebinding of von Willebrand factor with platelets functions as ananti-thrombosis drug which is efficacious in vivo such as in experimentswith animals, it is necessary to obtain a new active peptide prepared byconversion into a molecule which does not cause decrease in platelets.

DISCLOSURE OF THE INVENTION

The present invention has been made from the viewpoint described above,an object of which is to provide a peptide which inhibits the binding ofvon Willebrand factor with platelets without causing the decrease inplatelets, and a method of producing the peptide, in order to obtain adrug which is efficacious as an anti-thrombosis drug.

As a result of diligent studies in order to achieve the object describedabove, the present inventors have found that a single strand peptide,which is obtained by dissociating a multimer peptide from a snake venomunder a certain condition, has an anti-thrombus activity without causingany decrease in platelets upon in vivo administration. Thus the presentinvention has been completed.

Namely, the present invention lies in a single strand peptide obtainedby disconnecting disulfide bonds between peptide chains for constitutinga multimer peptide from a snake venom having an activity to inhibitbinding between von Willebrand factor and platelets while substantiallypreserving disulfide bonds within said peptide chains, wherein thesingle strand peptide (hereinafter referred to as "peptide of thepresent invention" or "active peptide", if necessary) does notsubstantially cause decrease in platelets at a minimum dose forexhibiting the activity in vivo.

In another aspect, the present invention provides a method of producinga single strand peptide obtainable from a multimer peptide from a snakevenom having an activity to inhibit binding between von Willebrandfactor and platelets, wherein the single strand peptide does notsubstantially cause decrease in platelets at a minimum dose forexhibiting the activity in vivo, the method comprising the steps ofallowing the multimer peptide from the snake venom to exist togetherwith a protein-denaturing agent, glutathione and/or cysteine, andthereby disconnecting disulfide bonds between peptide chains forconstituting the multimer peptide while substantially preservingdisulfide bonds within the peptide chains.

In still another aspect, the present invention provides a method ofproducing a peptide which does not substantially cause decrease inplatelets at a minimum dose for exhibiting an activity to inhibitbinding between von Willebrand factor and platelets in vivo, the methodcomprising the steps of cultivating, in an appropriate medium,Escherichia coli transformed with a vector containing an inserted DNAfragment coding for the peptide or a part thereof, expressing thepeptide, allowing the peptide accumulated in cells of Escherichia colito exist together with a protein-denaturing agent, and then generatingdisulfide bonds within chains of the peptide by removing theprotein-denaturing agent or by decreasing concentration of theprotein-denaturing agent.

In still another aspect, the present invention provides a method ofproducing a peptide which does not substantially cause decrease inplatelets at a minimum dose for exhibiting an activity to inhibitbinding between von Willebrand factor and platelets in vivo, the methodcomprising the steps of cultivating, in an appropriate medium, culturedinsect cells or cultured animal cells transformed with a vectorcontaining an inserted DNA fragment coding for the peptide or a partthereof, expressing the peptide, and recovering the peptide accumulatedin the cells or in the medium.

In still another aspect, the present invention provides a pharmaceuticalcomposition containing an efficacious component of the peptide and/or apharmaceutically acceptable salt thereof.

It is noted that the term "peptide" simply referred to in thisspecification indicates the single strand peptide in some cases, whilethe term indicates the multimer peptide in other cases.

The present invention will be described in detail below.

<1> The Peptide of the Present Invention

The peptide of the present invention is a single strand peptide obtainedby disconnecting disulfide bonds between peptide chains for constitutingthe multimer peptide from a snake venom having the activity to inhibitthe binding between von Willebrand factor and platelets whilesubstantially preserving disulfide bonds within said peptide chains. Thepeptide of the present invention is further characterized in that itdoes not substantially exhibit the decrease in platelets or thrombocytes(thrombocytopenia) at a minimum dose for exhibiting the activity invivo.

The present invention is applicable to any multimer peptide from thesnake venom provided that the multimer peptide has the activity toinhibit the binding between von Willebrand factor and platelets. Themultimer peptide includes, for example, peptides originating from snakevenoms produced by Crotalus horridus horridus, Cerastes cerastes, Echiscarinatus, Trimeresurus albolabris, and Vipera palaestina. Among them, apeptide from a snake venom produced by Crotalus horridus horridus ispreferred.

Specifically, the peptide of the present invention is represented by asingle strand peptide obtained from the snake venom peptide originatingfrom Crotalus horridus horridus, including those having an amino acidsequence shown in SEQ ID NO: 1 in Sequence Listing at their N-terminals.The peptide of the present invention also includes peptides having anamino acid sequence shown in SEQ ID NO: 2 in Sequence Listing, andcomprising disulfide bonds between 4th and 15th cysteine residues,between 32th and 120th cysteine residues, and between 95th and 112thcysteine residues as counted from the N-terminal in SEQ ID NO: 2. Thepeptide can be also produced by using genetic engineering techniques. Insuch production, a methionine residue corresponding to a translationinitiation codon is occasionally added to the N-terminal of the aminoacid sequence shown in SEQ ID NO: 1 or 2 in Sequence Listing. Thepeptide of the present invention also includes peptides with themethionine residue added at the N-terminal as described above.

It is also possible to produce peptides which have the activitydescribed above and have the amino acid sequence shown in SEQ ID NO: 2in Sequence Listing or a part thereof by using, for example, Escherichiacoli, cultured insect cells, or cultured animal cells in accordance withknown genetic engineering techniques. When the peptide of the presentinvention is expressed by using Escherichia coli as a host, inclusionbodies are formed in cells. However, a peptide having the activity isobtained by solubilizing the inclusion bodies, and forming disulfidebonds correctly. In such a procedure, any cysteine residue, which doesnot participate in formation of the disulfide bond, may be substitutedwith an amino acid other than cysteine such as alanine or serine. Thusit is expected to avoid occurrence of erroneous disulfide bondformation, and improve the stability of an obtainable peptide. The aminoacid sequence shown in SEQ ID NO: 2 includes amino acids or regionswhich are not necessary for the activity. It is also possible toconstruct peptides with substitution, deletion, or insertion at such oneor more amino acids. For example, the activity is maintained even when15 amino acid residues or 65 amino acid residues at the N-terminaland/or 11 amino acid residues at the C-terminal are deleted from thepeptide having the amino acid sequence shown in SEQ ID NO: 2, asdemonstrated in Example 6 described below.

Site-directed nucleotide sequence mutants can be prepared by using acommercially available kit (for example, Mutan-G and Mutan-K produced byTakara Shuzo). Alternatively, mutants can be also obtained by utilizingthe PCR process as described in "PCR protocols" (published by AcademicPress).

The single-stranded peptide of the present invention as described abovedoes not substantially cause the decrease in platelets which would becaused by the multimer peptide, while maintaining the activity toinhibit the binding between von Willebrand factor and platelets. Thepeptide of the present invention having such properties exhibits theanti-thrombus activity in vivo, and it is useful as an anti-thrombosisdrug.

<2> Method of Producing the Peptide of the Present Invention

For example, the following method is conceivable as one of means forobtaining, from the snake venom peptide, the peptide which does notcause the decrease in platelets while maintaining the activity toinhibit the binding between von Willebrand factor and platelets.

Assuming that the decrease in platelets upon administration of the snakevenom peptide, which occurs in vivo, results from, among other things, acause of the lectin activity of the snake venom peptide capable ofappearing in the presence of calcium, it is considered that the activityto cross-link platelets disappears by separating sites from each otherwhich correspond to sugar-binding sites existing in the lectin molecule.In order to separate such sugar-binding sites, for example, the multimerpeptide may be disconnected into individual single strand chains.

In order to divide the multimer peptide into single strand chains, thereare well-known methods including, for example, reduction of multimerpeptides, or reduction of multimer peptides followed by protection andstabilization of free thiol groups of cysteine residues by means ofcarboxymethylation, carboxyamidomethylation, or pyridylethylation.However, in general, water-soluble proteins have a higher-orderstructure in which side chains of major hydrophilic amino acid residuesare directed to the outside. Such a tertiary structure is destroyed by astrong denaturing condition, and a secondary structure is destroyed byreduction to cleave disulfide bonds between cysteine residues, or byprotection of free thiol groups of cysteine residues after reduction,sometimes resulting in insolubilization in water. As demonstrated inExample 1 described below, a double strand peptide obtained fromCrotalus horridus horridus was changed into a peptide scarcely solublein water on account of reducing carboxyamidomethylation, reducingpyridylmethylation, reduction with mercaptoethanol, etc.

Peng et al. (M. Peng et al., Blood, 81, 2321-2328 (1993)) has reportedthat a reaction mixture prepared by reduction of a double strand peptideobtained from Echis carinatus followed by carboxyamidomethylation alsohas a platelet aggregation-inhibiting activity similar to that of thedouble strand peptide. However, it is not reported at all whether or notthe inhibition on aggregation is specific, or what produced moleculeexhibits the activity.

Accordingly, in the present invention, the peptide from a snake venom isgently reduced under a mild denaturing condition without causing seriousdestruction of its higher-order structure. Thus disulfide bonds in thesingle strand chain of the double strand peptide, i.e. intramoleculardisulfide bonds are substantially preserved, while disulfide bondsbetween the chains, i.e. intermolecular disulfide bonds aredisconnected. Consequently, the single strand peptide is obtained whichis highly water-soluble, and maintains the original higher-orderstructure at least at an extent that the peptide does not lose theactivity.

In the present invention, the active peptide can be produced by usingthe multimer peptide contained in a snake venom which inhibits thebinding of von Willebrand factor with platelets. For example, the activepeptide is produced from a venom of Crotalus horridus horridus, or froma peptide obtained from a lyophilized product of the venom. It is notedthat the inhibition on the binding of von Willebrand factor withplatelets can be determined on the basis of inhibition on plateletaggregation induced by ristocetin or botrocetin (hereinafter simplyreferred to as "ristocetin-induced aggregation" or "botrocetin-inducedaggregation", if necessary.

Means for disconnecting disulfide bonds between peptide chains toprovide single strand chains while maintaining disulfide bonds betweencysteine residues within the peptide chain include a method in which themultimer peptide is reacted by allowing it to exist together with aprotein-denaturing agent, glutathione and/or cysteine. Guanidinehydrochloride, urea, etc. are used as the protein-denaturing agent. Thefinal concentration of guanidine hydrochloride is from 0.01M to asaturated concentration. Preferably, guanidine hydrochloride is used ina concentration range of 1M to 6M. Glutathione and/or cysteine is usedto perform reduction under a mild condition. When they are used, theyare appropriately added in a concentration range of 0.1 mM to 100 mM,preferably 1 mM to 50 mM.

The reaction is performed in a buffer containing Tris-salt, phosphatesalt, acetate salt, etc., in distilled water, or in a solution preparedby adding organic solvent such as alcohol to any of them. The reactionis performed in a state of solution in a temperature range of -10° C. to100° C., preferably 10 ° C. to 40° C. Thus the objective single strandpeptide can be obtained. The single strand peptide can be also obtainedby performing a freezing and thawing treatment.

The active peptide produced as described above can be isolated bycombining various methods such as gel filtration, ion exchange,adsorption, and reverse phase column chromatography, affinity columnchromatography, ultrafiltration, electrophoresis, and countercurrentdistribution.

Alternatively, the peptide of the present invention can be produced byusing microorganisms or cultured cells by utilizing geneticrecombination techniques for handling genes coding for the peptide. Thegene coding for the peptide of the present invention is obtained byscreening based on hybridization with a DNA fragment deduced from a partof the amino acid sequence for searching through a cDNA library obtainedfrom a venom gland of Crotalus horridus horridus or a genomic DNAlibrary obtained from a tissue, or by screening based on expressedproteins obtained from transformed cells allowed to express DNA includedin the library, the transformed cells including, for example,prokaryotes represented by Escherichia coli, fungi such as yeast, andcultured cells such as those of insects and animals. It is also possibleto combine and synthesize DNA sequences designed by using codonscorresponding to respective amino acids, and appropriate regulatorsequences, with reference to the amino acid sequence of the peptide ofthe present invention.

A clone having cDNA coding for the peptide of the present invention canbe selected, for example, by extracting total RNA from a venom gland ofCrotalus horridus horridus, purifying a mRNA fraction, synthesizing cDNAby using the mRNA as a template, preparing a cDNA library by using phageor the like, and conducting hybridization by using the cDNA library withan oligonucleotide probe prepared on the basis of the amino acidsequence of the peptide of the present invention. Alternatively, it isalso available to use, as a probe for hybridization, a DNA fragmentamplified from mRNA in accordance with the RT-PCR method by usingoligonucleotide primers prepared on the basis of the amino acid sequenceof the peptide of the present invention.

E. coli HB101/pCHA1 (E. coli AJ13023), which harbors a plasmid pCHA1containing a gene coding for the peptide of the present inventionobtained in Example described below, has been internationally depositedunder a deposition number of FERM BP-4781 based on the Budapest Treatysince Aug. 12, 1994 in National Institute of Bioscience and HumanTechnology of Agency of Industrial Science and Technology of Ministry ofInternational Trade and Industry (postal code: 305, 1-3 Higashi-Icchome,Tsukuba-shi, Ibaraki-ken, Japan).

The objective peptide can be expressed and produced under an appropriatecondition by inserting the obtained gene into a plasmid or virus vectorDNA containing a promoter, a translation initiation signal and so onexpressible in a host, and performing introduction of the plasmid, orinfection with the virus with respect to prokaryotes represented byEscherichia coli, fungi such as yeast, and cultured cells such as thoseof insects and animals. In this embodiment, the objective peptide can beaccumulated in microbial cells or cultured cells, or it can be producedand secreted into a culture liquid. The objective peptide can bedirectly expressed in a form in which methionine as a translationinitiation codon is added thereto. Alternatively, the objective peptidecan be expressed in a form in which a secretion signal sequence isadded. The objective peptide can be produced by cleaving and removingthe signal sequence during secretion process. In another embodiment, theobjective peptide may be expressed as a chimeric protein fused withanother appropriate protein (for example, Escherichia coli maltosebinding protein). In such an embodiment, the objective protein can beobtained by cleavage with an appropriate protease or with a chemicalmethod after expressing the chimeric protein. When the protein thusexpressed and produced does not have the objective activity due to astate of its higher-order structure, or when the protein only has a weakactivity, it can be changed to form a higher-order structure having asufficient activity by means of a treatment under an appropriatedenaturing condition or under an appropriate oxidation-reductioncondition.

When the peptide of the present invention is produced by usingEscherichia coli as a host, the produced peptide forms inclusion bodiesin cells as described above. However, the peptide having the activitycan be obtained by solubilizing the inclusion bodies, and formingdisulfide bonds correctly. Specifically, the peptide of the presentinvention is obtained by cultivating, in an appropriate medium,Escherichia coli transformed with a vector containing an inserted DNAfragment coding for the peptide of the present invention or a partthereof, expressing the peptide, allowing the peptide accumulated incells of Escherichia coli to exist together with a protein-denaturingagent, and then generating disulfide bonds within chains of the peptideby removing the protein-denaturing agent or by decreasing concentrationof the protein-denaturing agent. The disulfide bonds are formed, forexample, between 4th and 15th cysteine residues, between 32th and 120thcysteine residues, and between 95th and 112th cysteine residues in SEQID NO: 2 when the peptide has the amino acid sequence shown in SEQ IDNO: 2.

Alternatively, the peptide of the present invention is obtained bycultivating, in an appropriate medium, cultured insect cells or culturedanimal cells transformed with a vector containing an inserted DNAfragment coding for the peptide or a part thereof, expressing thepeptide, and recovering the peptide accumulated in the cells or in themedium.

<3> Pharmaceutical Composition of the Present Invention

The peptide obtained as described above does not cause the decrease inplatelets even after administration to animals. In fact, it remarkablyinhibited thrombus formation after administration to thrombosis modelanimals.

Except for the dye substance such as Aurin tricarboxylic acid which hasthe property to non-specifically adsorb to proteins, the presentinvention has disclosed the substance for the first time that exhibitsthe anti-thrombus activity in vivo based on the feature that the bindingof von Willebrand factor with platelets is inhibited. Namely, thepresent invention has revealed the fact for the first time that theactivity to inhibit the binding of von Willebrand factor with plateletsexists in the single strand peptide obtained from the peptideoriginating from a snake venom by using the method such as reduction.Thus the present invention provides a pharmaceutical composition whichis extremely hopeful as an anti-thrombosis drug that does not cause thedecrease in platelets upon administration in vivo.

The pharmaceutical composition of the present invention contains thepeptide of the present invention and/or a pharmaceutically acceptablesalt thereof as an active ingredient. A mixture of one or more speciesof the peptides of the present invention may be used. In one embodiment,the composition may contain substances having any anti-thrombosisfunction other than the peptide of the present invention. In such anembodiment, the peptide of the present invention is not necessarily amajor component of the pharmaceutical composition. The composition maybe blended with other materials ordinarily used as components for drugpreparation including, for example, proteins such as serum albumin,salts for buffering action or osmotic pressure adjustment, carriers, andexcipients.

The type of drug includes, for example, tablet, capsule, granule, syrup,suppository, ointment, injection, and instillation. Among them,injection drugs are preferred. The method of administration may be anyof intravenous, subcutaneous, oral, ophthalmic, transintestinaladministrations, etc. Among them, intravenous administration ispreferred.

As for the dose upon administration to animals or human, an intendedeffect can be usually expected in a range of 0.1 μg/kg to 100 mg/kg, asan amount of the peptide of the present invention and/or thepharmaceutically accepted salt thereof. Within this range, it ispossible to select an amount with which the most excellent medicinaleffect is obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reverse phase chromatogram of CH-1.

FIG. 2 shows a reverse phase chromatogram of CH-2.

FIG. 3 shows a reverse phase chromatogram of CH-1 before addition ofglutathione.

FIG. 4 shows a reverse phase chromatogram of substances produced fromCH-1, 5 days after addition of glutathione.

FIG. 5 shows a reverse phase chromatogram of substances produced fromCH-1.

FIG. 6 shows production amounts (μg/tube) of (a) Peak 1 and (b) Peak 2under respective conditions depending on the passage of time.

FIG. 7 shows an amino acid sequence of AS1051. Fragments A, B, C wereobtained as one fragment connected through disulfide bonds bylysylendopeptidase digestion.

FIG. 8 shows inhibiting activities (inhibition ratio with respect tocontrol) of (a) CH-1 and (b) AS1051 on ristocetin-mediated aggregationand botrocetin-mediated aggregation.

FIG. 9 shows inhibiting activities of AS1051 on ristocetin-mediatedaggregation and botrocetin-mediated aggregation of guinea pig platelets.

FIG. 10 shows aggregating activities of (a) ristocetin and (b)botrocetin on guinea pig platelets after administration of AS1051 (200μg/kg).

FIG. 11 shows an anti-thrombus activity of AS1051 (administrationamount: 200 μg/kg) on optically excited thrombus model guinea pigs.

FIG. 12 shows construction steps for an AS1051 expression plasmid pCAHT7for Escherichia coli.

FIG. 13 shows construction steps for an AS1051 expression plasmidpCHAbac for cultured insect cells.

FIG. 14 shows an inhibiting activity of AS1051 in a cell-disruptedsolution expressed by cultured insect cells on binding of von Willebrandfactor with fixed platelets evoked by botrocetin.

FIG. 15 shows an inhibiting activity of AS1051 in a culture supernatantexpressed by cultured insect cells on binding of von Willebrand factorwith fixed platelets evoked by botrocetin.

FIG. 16 shows construction steps for an AS1051 expression plasmidpCHASDX for cultured animal cells (CHO cells).

FIG. 17 shows an inhibiting activity of AS1051 in a culture supernatantexpressed by CHO cells on binding of von Willebrand factor with fixedplatelets evoked by ristocetin.

FIG. 18 shows an inhibiting activity of AS1051 in a culture supernatantexpressed by CHO cells on binding of von Willebrand factor with fixedplatelets evoked by botrocetin.

FIG. 19 shows construction steps for a plasmid pCHAT7Ala for expressing,in Escherichia coli, AS1051 having mutation to substitute an alanineresidue for an 81th cysteine residue as counted from the N-terminal(except for methionine residue for translation initiation).

FIG. 20 shows inhibiting activities of AS1051 prepared from a snakevenom, AS1051 produced by Escherichia coli, and AS1051 (Cys81Ala mutantAS1051) with an alanine residue substituted for an 81th cysteine residueas counted from the N-terminal, on binding of von Willebrand factor withfixed platelets evoked by ristocetin or botrocetin.

FIG. 21 schematically shows structures of various shortened AS1051peptides.

FIG. 22 shows an HPLC chromatogram of AS1051A-1.

FIG. 23 shows an HPLC chromatogram of AS1051A-2.

FIG. 24 shows an HPLC chromatogram of AS1051A-3.

FIG. 25 shows an HPLC chromatogram of AS1051A-4.

FIG. 26 shows an HPLC chromatogram of AS1051A-5.

FIG. 27 shows inhibiting activities of the various shortened AS1051peptides on binding of von Willebrand factor with fixed platelets evokedby ristocetin or botrocetin.

BEST MODE FOR CARRYING OUT THE INVENTION

Examples of the present invention will be described below.

EXAMPLE 1 Preparation of Anti-Thrombus Single Strand Peptide fromCrotalus horridus horridus

<1> Preparation of Peptide Having Activity to Inhibit Binding of vonWillebrand Factor with Platelets from Crotalus horridus horridus

A peptide having an activity to inhibit binding of von Willebrand factorwith platelets was purified from a snake venom originating from Crotalushorridus horridus by using an index of an activity to inhibitristocetin-mediated aggregation in accordance with a method describedbelow.

Fresh blood collected from healthy human added with 1/10 volume of 3.8%sodium citrate was centrifuged at 900 rpm for 15 hours to obtain humanplatelet rich plasma to which an equal volume of physiological salinesolution containing 2% paraformaldehyde was added, followed by beingstored at 4° C. overnight stationarily. After the storage, plateletswere recovered by centrifugation, and they were washed twice with aphysiological saline solution containing 20 mM phosphate buffer (pH7.4). A sample for measurement was added to a formalin-fixed plateletsolution thus prepared, to which human plasma (final concentration:0.12%) and ristocetin sulfate (produced by Sigma, final concentration:0.5 mg/ml) were successively added. After shaking and agitation, thepresence or absence of the inhibiting activity on platelet aggregationwas observed macroscopically. The reaction solution had a volume of 50μl. The sample was properly diluted, and added in an appropriate amountof 2 to 20 μl.

A lyophilized snake venom product (1 g) from Crotalus horridus horridus(produced by Sigma) was dissolved in a physiological saline solution (10ml) containing 20 mM Tris-HCl (pH 7.4). Insoluble matters were removedby centrifugation at 3,000 rpm for 10 minutes. A supernatant wassubjected to gel filtration chromatography by using the same buffer as asolvent and using a Sephadex G-75 (fine) (produced by Pharmacia) column(diameter: 5.0 cm, length: 90 cm) to make fractionation into fractiontubes. A fraction corresponding to an elution volume from 750 ml to 885ml was collected, and divided into aliquots. Three aliquots, each havinga volume of 15 ml, were allowed to pass through a benzamidine Sepharose(produced by Pharmacia) column (diameter 1.6 cm, length 5 cm) to collecta non-adsorbed fraction.

This fraction was filtrated and concentrated by using an ultrafiltrationmembrane (YM10, produced by Amicon) having en exclusion molecular weightof 10,000, and the concentrated fraction was solvent-substituted with a50 mM ammonium acetate buffer (pH 4.5). The fraction was adsorbed to anion exchange chromatography column (diameter 2.6 cm, length 30 cm) usingCM Sepharose CL6B (produced by Pharmacia) equilibrated with the samesolvent. The column was washed with the initial solvent for 50 minutes,followed by elution with a linear concentration gradient (810 minutes)from a solution containing 15% to 50% of a 0.5M ammonium acetate buffer(pH 6.4) mixed in the initial solvent. An eluted fraction correspondingto an elution volume from 610 ml to 650 ml was fractionated andcollected, followed by concentration by using the ultrafiltrationmembrane in the same manner as described above. After that, the solventwas substituted with a physiological saline solution containing 20 mMTris-HCl (pH 7.4). A similar platelet aggregation-inhibiting activitywas also present in an eluted fraction corresponding to an elutionvolume from 540 ml to 580 ml. Accordingly, this fraction was treated inthe same manner as described above.

Parts of the respective platelet aggregation-inhibiting activityfractions obtained by the ion exchange column chromatography wereapplied to high-performance liquid chromatography by using a reversephase column (SSC-VP-304, produced by Senshu Kagaku, diameter: 4.6 mm,length: 250 mm). Analysis was performed by elution with a linearconcentration gradient (20 minutes) from an acetonitrile concentrationof 31% to a concentration of 52% containing 0.1% trifluoroacetic acid.As a result, the both fractions contained single peptides which weredifferent with each other (FIGS. 1, 2). According to SDS-polyacrylamidegel electrophoresis, both of the peptides contained in the two activefractions presented one band having a molecular weight of about 25kilodaltons under a non-reduced condition, and two bands havingmolecular weights of about 14 kilodaltons and 15 kilodaltons under areduced condition obtained by adding 1% mercaptoethanol. It was foundthat the both are similar double strand peptides (SEQ ID NOS: 1 and 3).Accordingly, the peptide eluted in the latter active fractions from theion exchange column (elution volume: 610 ml to 650 ml) was designated asCH-1, and the peptide eluted in the former active fractions (elutionvolume: 540 ml to 580 ml) was designated as CH-2.

Amino acid sequence analysis for an amino terminus of CH-1 was performedas follows. About 50 μg of CH-1 was dissolved in 100 μl of a 7Mguanidine hydrochloride aqueous solution containing 0.5M Tris-HCl (pH8.5) and 10 mM ethylenediaminetetraacetic acid disodium salt (EDTAsNa2). After that, 4-vinylpyridine (1 μl) and tri-n-butylphosphine (2 μl)were added. A reaction was performed at room temperature overnight tocarry out reducing pyridylethylation. The reaction solution was appliedto high performance liquid chromatography by using a column ofTSKgel-Phenyl-5PW-RP (produced by Tosoh Co., diameter: 4.6 mm, length:75 mm) to separate each of produced peptide chains. Elution wasperformed at a flow rate of 1 ml/min with a linear concentrationgradient (20 minutes) from an acetonitrile concentration of 31% to aconcentration of 52% containing 0.1% trifluoroacetic acid. Each of theseparated peptide chains was lyophilized, and then dissolved in 30%acetonitrile to perform amino acid sequence analysis from the aminoterminus by using Protein Sequencer 470A (produced by AppliedBiosystems). As a result, it was found that the amino acid sequences onthe amino terminus of the respective chains were those shown in SEQ IDNO: 1 and 3 in Sequence Listing. They were respectively coincident withamino acid sequences on N-terminal sides of α-chain and β-chain of apeptide CHH--B isolated from the same snake venom as described in aninternational publication pamphlet of WO9208470. According to retentiontimes of CH-2 and CH-1 in the analysis by using the reverse phase columnchromatography shown in FIGS. 1 and 2, it was considered that CH-2 wassimilar to CHH-A, and CH-1 was similar to CHH-B described in thepamphlet.

<2> Activity of CH-1 upon Administration to Animal

The number of platelets was measured when CH-1 obtained as describedabove was administrated to guinea pigs. CH-1 was dissolved in aphysiological saline solution to give concentrations of 10 to 100 μg/ml,and administrated intravenously to guinea pigs. After about 5 minutes,arterial blood was collected from abdominal aorta. Citric acid (0.32%)was used as a blood anticoagulant in the blood collection to measure thenumber of white blood corpuscles (WBC), the number of red bloodcorpuscles (RBC), and the number of platelets (PLT) by using SysmexE-2000 (produced by Toa Medical Electronics).

Administration doses and the numbers of the respective corpuscles areshown in Table 1. Only the decrease in platelets was observed dependingon the dose of administration. Almost complete disappearance ofplatelets was observed at an administration dose of 100 μg/kg or more.

                  TABLE 1    ______________________________________    Measurement              Physiological                         CH-1 (μg/kg)    item      saline     10     30   30    100  100    ______________________________________    WBC × 10.sup.2 /μl              35.0       18.0   20.0 19.0  19.0 21.0    RBC × 10.sup.4 /μl              465.0      440.0  440.0                                     457.0 468.0                                                448.0    PLT × 10.sup.4 /μl              38.3       41.0   9.6  13.7  0.4  0.8    ______________________________________

<3> Trial to Convert Double Strand CH-1 into Single Strand

(1) Reducing Carboxyamidomethylation for CH-1

Reducing carboxyamidomethylation for CH-1 was performed as follows. CH-1(200 μg) was dissolved in 500 μl of a 7M guanidine hydrochloride aqueoussolution containing 0.5M Tris-HCl (pH 8.5) and 10 mMethylenediaminetetraacetic acid disodium salt (EDTA·Na2), to which 50 μlof a dithiothreitol aqueous solution (20 mg/ml) was added, followed bystorage at 37° C. for 1 hour. An iodoacetoamide aqueous solution (50 μl,50 mg/ml) was added to the stored solution to perform a reaction at roomtemperature for 30 minutes with shielding from light.

After the reaction, dialysis was performed at 4° C. overnight against a0.15M sodium chloride aqueous solution (5 liters) containing 20 mMTris-HCl (pH 7.4) by using a dialysis membrane Spectra/por1 (produced bySpectra, dialysis limit molecular weight: 6,000 to 8,000). Since whiteinsoluble matters appeared after the dialysis, a surfactant of Tween-20was added to give a final concentration of 2%. However, the insolublematters were not dissolved. The white-turbid solution was concentratedby ultrafiltration by using Centricon-10 (produced by Amicon), and asuspension of 100 μl was finally obtained. The suspension was weaklycentrifuged to obtain a supernatant. After that, an aliquot (10 μl) ofthe supernatant was used to investigate the inhibiting activity onristocetin-mediated aggregation in accordance with the method describedin the item <1>. However, the inhibiting activity was not found. It wasconsidered that this result was obtained because CH-1 subjected toreducing carboxyamidomethylation by the method described above had noinhibiting activity on ristocetin-mediated aggregation, or because itconsequently had an extremely low solubility in water.

(2) Reducing Pyridylethylation for CH-1

Reducing pyridylethylation for CH-1 was performed as follows. CH-1 (200μg) was dissolved in 200 μl of a 7M guanidine hydrochloride aqueoussolution containing 0.5M Tris-HCl (pH 8.5) and 10 mMethylenediaminetetraacetic acid disodium salt (EDTA·Na2), to which4-vinylpyridine (1 μl) and tri-n-butylphosphine (2 μl) were added toperform a reaction at room temperature overnight. The reaction solutionwas applied to high-performance liquid chromatography by using a reversephase column (SSC-VP-304, produced by Senshu Kagaku, diameter: 4.6 mm,length: 250 mm). Elution was performed with a concentration gradient (20minutes) from an acetonitrile concentration of 10% to a concentration of59% containing 0.1% trifluoroacetic acid. Products were fractionated andcollected as a mixture of two species of pyridylethylated peptides.

The fractionated fraction was lyophilized, and then the total amount wasdissolved in 50 μl of a physiological saline solution containing 20 mMTris-HCl (pH 7.4). An aliquot (10 μl) was used to observe the inhibitingactivity on ristocetin-mediated aggregation in accordance with themethod described in the item <1>. However, the inhibiting activity wasnot observed. It was considered that this result was obtained becauseCH-1 subjected to reducing pyridylethylation by the method describedabove had no inhibiting activity on ristocetin-mediated aggregation, orbecause it consequently had an extremely low solubility in water.

(3) Reduction of CH-1 with Mercaptoethanol and Reconstruction ofDisulfide Bonds

Operations were performed as follows for reduction of CH-1 withmercaptoethanol and reconstruction of disulfide bonds after thereduction with redox (oxidation-reduction) buffers by using reduced andoxidized glutathione.

CH-1 (240 μg) was dissolved in 120 μl of a 7M guanidine hydrochlorideaqueous solution containing 0.5M Tris-HCl (pH 8.5) and 10 mMethylenediaminetetraacetic acid disodium salt (EDTA·Na2), which wasstored at 37° C. for 10 minutes. A 10% mercaptoethanol aqueous solution(1/9 volume) was added to the solution to give a final concentration ofmercaptoethanol of 1%, followed by being stored to stand at 37° C. for 1hour. After that, 2 ml of a solution (concentration of guanidinehydrochloride: 2M, hereinafter abbreviated as "2M guanidinehydrochloride preparation solution"), which was obtained by diluting the7M guanidine hydrochloride aqueous solution containing 0.5M Tris-HCl (pH8.5) and 10 mM ethylenediaminetetraacetic acid disodium salt (EDTA·Na2)by a factor of 2/7, was added, followed by ultrafiltration concentrationby using Centricon-10 (produced by Amicon). The 2M guanidinehydrochloride preparation solution (2 ml) was added to the obtainedconcentrated solution, followed by ultrafiltration concentration again.This concentrating operation was repeated several times to removemercaptoethanol. Thus a 2M guanidine hydrochloride preparation solution(380 μl) containing reduced products of CH-1 was obtained.

The solution containing reduced products of CH-1 was divided into 5aliquots (each having a volume of 76 μl) to perform an operation forreconstructing disulfide bonds with redox buffers by using reduced andoxidized glutathione. The 2M guanidine hydrochloride preparationsolution (374 μl) was added to each of the aliquots. Solutions (50 μl)obtained by mixing reduced and oxidized glutathione in ratios shown inTable 2 were further added respectively, followed by substitution withnitrogen. After that, the vessels were tightly sealed to perform thereaction at room temperature overnight.

                  TABLE 2    ______________________________________                   Buffer    Composition of redox buffer                     1       2     3     4   5    ______________________________________    Aqueous solution of                     1       2     1     2   4    50 mM oxidized glutathione    Aqueous solution of                     5       5     1     1   1    100 mM reduced glutathione    ______________________________________     (Numerical values indicate mixing ratios of the solutions.)

The five reaction solutions above and solutions immediately afterreduction with mercaptoethanol were applied to high-performance liquidchromatography by using a reverse phase column (SSC-VP-304, produced bySenshu Kagaku, diameter: 4.6 mm, length: 250 mm). Analysis was performedby monitoring absorbance at 216 nm while performing elution by using aconcentration gradient (20 minutes) from an acetonitrile concentrationof 10% to a concentration of 59% containing 0.1% trifluoroacetic acid.However, no peak originating from any produced peptide was observed inall of the solutions.

It was considered that the result described above was obtained becauseCH-1 was converted into a reduced product having low solubility as aresult of reduction with mercaptoethanol, and because the reducedproduct was not converted into a substance having high solubility bymeans of the oxidation-reduction reaction performed with the redoxbuffers.

(4) Reduction of CH-1 with Glutathione

A mild reducing reaction for CH-1 with glutathione was performed asfollows. CH-1 (40 μg) was dissolved in the 2M guanidine hydrochloridepreparation solution (450 μl) described above. After that, a solution(50 μl) of reduced glutathione (100 mM) dissolved in the samepreparation solution was added, followed by being left to stand at 40°C. for 3 hours, and followed by being left to stand at room temperaturefor 5 days. The feature of products was analyzed by high-performanceliquid chromatography by using a reverse phase column (SSC-VP318,produced by Senshu Kagaku, diameter: 4.6 mm, length: 250 mm). Elutionwas performed with a concentration gradient (20 minutes) from anacetonitrile concentration of 10% to a concentration of 80% containing0.1% trifluoroacetic acid to observe absorbance at 216 nm.

FIGS. 3 and 4 show chromatograms before addition of glutathione (FIG. 3)and 5 days after addition of glutathione (FIG. 4). The total amount ofthe reaction solution after the reducing reaction with glutathione wasseparated by the same chromatography to fractionate and collect Peaks 1,2, 3 shown in FIG. 4 respectively. Each of the peaks was lyophilized,and then the total amount was dissolved in a 0.15M sodium chlorideaqueous solution (25 μl) containing 20 mM Tris-HCl (pH 7.4). An aliquot(10 μl) was used to measure the inhibiting activity onristocetin-mediated aggregation in accordance with the method describedin the item <1>. As a result, Peaks 1 and 3 had the inhibiting activity.However, Peak 3 was a peak of the raw material (CH-1). According to theresult described above, it was revealed that the new substance (Peak 1)was generated by the method described above, the new substance havingthe inhibiting activity on ristocetin-mediated aggregation.

The effect of glutathione at various concentrations was investigated inthe presence of 6M or 2M guanidine hydrochloride. CH-1 (36 μg) wasdissolved in 234 μl of a solution (hereinafter referred to as "6Mguanidine hydrochloride preparation solution") obtained by diluting a 7Mguanidine hydrochloride aqueous solution containing 0.5M Tris-HCl (pH8.5) and 10 mM ethylenediaminetetraacetic acid disodium salt (EDTA·Na2)by a factor of 6/7, or a solution (hereinafter referred to as "2Mguanidine hydrochloride preparation solution") obtained by diluting the7M guanidine hydrochloride aqueous solution by a factor of 2/7. To thesolutions obtained by dissolving CH-1 in the 6M and 2M guanidinehydrochloride preparation solutions glutathion solutions (26 μl) havingrespective 10-fold concentrations were added so that the finalconcentration of reduced glutathione was 30 mM, 10 mM, 3 mM, and 1 mMrespectively, and that the final concentration of oxidized glutathionewas 30 mM. The feature of produced peaks after 1, 2, 3, 4, and 7 dayswas analyzed by high-performance liquid chromatography by using areverse phase column (SSC-VP318, produced by Senshu Kagaku, diameter:4.6 mm, length: 250 mm). Elution was performed with a concentrationgradient (20 minutes) from an acetonitrile concentration of 24% to aconcentration of 59% containing 0.1% trifluoroacetic acid monitoringabsorbance at 216 nm.

FIG. 5 shows one example of chromatogram obtained by this analysis (2Mguanidine hydrochloride, 10 mM reduced glutathione, 4 days). Owing tothis analysis system, it was found that those eluted at a retention timecorresponding to Peak 1 shown in FIG. 4 contained two species ofsubstances (corresponding to Peaks 1, 2 shown in FIG. 5). Peak 4 was apeak of the raw material (CH-1). Produced amounts of the substancescontained in Peak 1 and Peak 2 changed depending on time underrespective reaction conditions as shown in FIG. 6.

<4> Production of Single Strand Peptide from CH-1 by Using ReducedGlutathione

Distilled water (7.39 ml), a 7M guanidine hydrochloride aqueous solutioncontaining 0.5M Tris-HCl (pH 8.5) and 10 mM ethylenediaminetetraaceticacid disodium salt (EDTA·Na2) (3.57 ml), and a 100 mM reducedglutathione (produced by Boehringer Mannheim) aqueous solution (1.25 ml)were added to a physiological saline aqueous solution of CH-1 (1.3mg/ml, 290 μl), followed by storage at 28° C. for 5 days (finalconcentration of guanidine hydrochloride: 2M).

The solution after the storage was centrifuged at 3,000 rpm, and itssupernatant was adjusted to be acidic (pH 4 or lower) by usingtrifluoroacetic acid. After that, the supernatant was applied to areverse phase column (Vydac 214TP1022, produced by Vydac, diameter: 22mm, length: 250 mm) at a flow rate of 15 ml/min to fractionate andcollect produced peptides in accordance with elution by using aconcentration gradient (20 minutes) from an acetonitrile concentrationof 27% to a concentration of 45% containing 0.1% trifluoroacetic acid.

As a result, the same products as those shown in FIG. 5 were generated.Peak 1 and Peak 2 were designated as AS1051 and AS1052 respectively. Asa result of SDS-polyacrylamide gel electrophoresis for each of them, itwas demonstrated that AS1051 was a single-stranded peptide having amolecular weight of about 14 kilodaltons under a non-reduced conditionand a molecular weight of about 15 kilodaltons under a reduced conditionin the presence of 1% mercaptoethanol. It was also suggested that AS1052was a homodimer comprising the same two peptides having molecularweights of about 26 and 15 kilodaltons under non-reduced and reducedconditions respectively.

The amino acid sequence and the manner of disulfide bonds of obtainedAS1051 were determined as follows. A 1M Tris-HCl buffer (20 μl)containing 20 mM ethylenediaminetetraacetic acid disodium salt (EDTA),distilled water (120 μl), an aqueous solution (10 μl) containing 5 μg oflysylendopeptidase (produced by Wako Pure Chemical) were successivelyadded to an aqueous solution (50 μl) containing 50 μg of AS1051 toperform an enzymatic digestion reaction at 37° C. for 2 hours. Thereaction solution was applied to high-performance liquid chromatographyby using a reverse phase column (SSC-VP-318, produced by Senshu Kagaku,diameter: 4.6 mm, length: 250 mm) to separate, fractionate and collectdigested fragments. After that, each of the digested fragments wassubjected to amino acid sequence analysis by using Protein Sequencer470A (produced by Applied Biosystems). Thus the amino acid sequence ofAS1051 was determined as shown in SEQ ID NO: 2 and FIG. 7. In thissequence, the number of amino acid residues was smaller by one, and39th, 85th, and 86th amino acids were different, as compared withα-chain of CHH-B described in the international publication pamphlet ofWO9208472.

Three Fragments A, B, C shown in FIG. 7 were obtained as one digestedfragment bound through disulfide bonds. Accordingly, the fragment wasfurther digested with proteinase Glu-C (produced by BoehringerMannheim). As a result of amino acid sequence analysis for producedfragments, it was found that the disulfide bonds were formed betweenCys4 and Cys15, between Cys32 and Cys120, and between Cys95 and Cys112.This bonding manner is common to C-type lectin including thoseoriginating from snake venom. Accordingly, it is considered that AS1051maintains the manner of original disulfide bonds.

As a result of amino acid sequence analysis, it was found that doublestrand AS1052 was a homodimer comprising two chains of AS1051.

<5> Comparison of Platelet Aggregation-inhibiting Activities by UsingDouble Strand Peptide (CH-1) and Single-Sstranded Peptide (AS1051)

The inhibiting activity of the double strand CH-1 on plateletaggregation was compared with that of the single-stranded peptideAS1051. Fresh blood collected from healthy human added with a 1/10volume of 3.8% sodium citrate was centrifuged at 900 rpm for 15 minutesto obtain human platelet rich plasma (PRP) to which these peptides wereadded to measure resulting platelet aggregation-inhibiting activities byusing Hematracer-801 (produced by Niko Bioscience). The measurement wasperformed by adding 100 μl of the platelet rich plasma into a cuvettecontaining 12.5 μl of a peptide solution, agitating it at 37° C. for 3minutes by using a stirrer bar, and then adding 12.5 μl of anagglutinogen to observe transmitted light.

Ristocetin (final concentration: 1.2 mg/ml), botrocetin (1 μg/ml), ADP(3 μM), or collagen (10 μg/ml) was used as the agglutinogen to calculatethe aggregation-inhibiting ratio with respect to a control group towhich no peptide sample was added. FIG. 8 shows inhibiting activities ofCH-1 and AS1051 on ristocetin-induced aggregation and botrocetin-inducedaggregation. Almost the same inhibiting activity was observed for theboth peptides (double strand peptide (CH-1), and single-stranded peptide(AS1051)) against ristocetin-induced aggregation and botrocetin-inducedaggregation. The both peptides exhibited no inhibition on aggregationinduced by ADP and collagen even at a concentration of 20 μg/ml,demonstrating that they specifically inhibited aggregation depending onthe binding between von Willebrand factor and glycoprotein Ib, such asthe ristocetin-induced aggregation and the botrocetin-inducedaggregation.

It was also revealed that the double strand AS1052 had approximately thesame inhibiting activity as that of AS1051.

<6> Comparison of Numbers of Platelets upon Administration of DoubleStrand Peptide (CH-1) and Single-stranded Peptide (AS1051) to Mice

Physiological saline solutions of CH-1 and AS1051 (100 μg/kg for each),and only a physiological saline solution as a control were intravenouslyadministrated to mice respectively. Blood was collected from heartsafter 5 minutes to measure the number of white blood corpuscles (WBC),the number of red blood corpuscles (RBC), and the number of platelets(PLT) in the same manner as in the item <2>.

As shown in Table 3, platelets disappeared almost completely in thegroup of CH-1 administration (100 μg/kg). On the contrary, the decreasein platelets was not observed in the group of AS1051 administration (100μg/kg). No change was observed in the number of white blood corpuscles(WBC) and the number of red blood corpuscles (RBC) both in the groups ofCH-1 and AS1051 administration as compared with the control group.According to the fact described above, it was confirmed that thesingle-stranded AS1051 did not cause the decrease in platelets which wasobserved upon administration of the double strand peptide CH-1.

                  TABLE 3    ______________________________________             Physio-    Measurement             logical   AS1051       CH-1    item     saline    (100 μg/kg)                                    (100 μg/kg)    ______________________________________    WBC × 10.sup.2 /μl             7.0    21.0   51.0 13.0 20.0 37.0 21.0 21.0    RBC × 10.sup.4 /μl             885.0  830.0  882.0                                871.0                                     866.0                                          864.0                                               921.0                                                    891.0    PLT × 10.sup.4 /μl             130.9  136.3  122.4                                107.7                                     119.7                                          1.8  1.0  0.9    ______________________________________

<7> Measurement of Anti-thrombus Activity of Single-stranded Peptide byUsing Guinea Pigs

At first, the measurement was performed for inhibiting activities of thesingle-stranded peptide AS1051 on ristocetin-induced aggregation andbotrocetin-induced aggregation of platelet rich plasma obtained fromguinea pigs. As shown in FIG. 9, the inhibiting activities of AS1051 onthe ristocetin-induced aggregation and the botrocetin-inducedaggregation were at approximately the same degrees as those of thevalues obtained for the platelet rich plasma from human (FIG. 8).

Next, the numbers of corpuscles such as platelets were measured afteradministration of AS1051 to guinea pigs. Further, an exo-vivo test wasperformed by using platelet rich plasma prepared from blood collectedafter administration of AS1051 to measure whether or not the aggregationwas inhibited. AS1051 (200 μg/kg) was intravenously administrated toguinea pigs. Arterial blood at 5 minutes after the administration wascollected to measure the number of white blood corpuscles (WBC), thenumber of red blood corpuscles (RBC), and the number of platelets (PLT)(Table 4) in the same manner as in the item <2>.

                  TABLE 4    ______________________________________    Measurement    Physiological                                AS1051    item           saline       (200 μg/kg)    ______________________________________    WBC × 10.sup.2 /μl                   35.0   19.0   22.0 24.0 21.0 27.0    RBC × 10.sup.4 /μl                   509.0  512.0  522.0                                      518.0                                           458.0                                                471.0    PLT × 10.sup.4 /μl                   35.9   46.3   30.7 34.6 33.7 40.0    ______________________________________

The same amount of bovine serum albumin (BSA) was added to theadministrated preparations of AS1051, and those administrated with onlyBSA were used as a control group. No change was observed in the numberof platelet, etc. in the AS1051-administrated group as compared with thecontrol group. Platelet rich plasma was prepared from the blood tomeasure the features of ristocetin-induced aggregation andbotrocetin-induced aggregation by using the same method as describedabove (FIG. 10). Observation was made by changing the amount of addedristocetin. It was demonstrated that the aggregation was almostcompletely inhibited in the AS1051-administrated group even at aristocetin concentration at which the control group completely causedthe aggregation. As for the botrocetin-induced aggregation, the sameresult was obtained.

According to the results described above, it was demonstrated thatAS1051 did not affect the number of platelets upon intravenousadministration to guinea pigs in an amount of 200 μg/kg in the samemanner as in the item <5> described above (administration to mice), andthat a sufficient concentration of AS1051 was maintained in blood toinhibit the ristocetin-induced aggregation and the botrocetin-inducedaggregation.

Next, the anti-thrombus activity of AS1051 was evaluated in vivo byusing an animal thrombosis model. An optically excited thrombus modelreported by Matsuno et al. (Matsuno et al., Blood and Circulation(Ketsueki-to-Jyunkan), 4, pp. 20-23 (1990)) was used as the thrombosismodel.

Carotid artery of each of guinea pigs was exfoliated to install aDoppler blood flow probe. A xenon lamp light source was provided at aposition spaced by a distance of about 5 mm upstream from the bloodvessel installed with the probe. A sample of AS1051 containing BSA (theamount of BSA was the same as the amount of AS1051) or a samplecontaining only BSA as a control was intravenously administrated toguinea pigs. After 5 minutes, a rose bengal solution (10 mg/kg) wasintravenously administrated. Simultaneously, a light beam of 540 nm wasradiated to damage blood vessel walls. Thus the time until stop of bloodflow was measured by using a pulse Doppler blood flow meter. As shown inFIG. 11, the time until stop of blood flow was clearly prolonged in theAS1051-administrated group (200 μg/kg) as compared with the controlgroup, demonstrating that AS1051 had the anti-thrombus activity(p<0.01).

EXAMPLE 2 Production of Anti-Thrombus Single Strand Peptide byEscherichia coli

In order to produce the peptide of the present invention by usinggenetic engineering techniques, a gene coding for the peptide having theactivity to inhibit the binding of von Willebrand factor with plateletswas isolated from Crotalus horridus horridus, and it was expressed inEscherichia coli.

<1> Preparation of cDNA Library of Crotalus horridus horridus

(1) Extraction of mRNA from Crotalus horridus horridus

A venom gland of Crotalus horridus horridus was excised. It wasimmediately frozen with liquid nitrogen, and stored until use. Thepoison gland (1.7 g) was disrupted with Polytron Homogenizer (producedby Kinematica) in 20 ml of an RNA-extracting solution (4M guanidiumisothiocyanate hydrochloride, 0.1M Tris-HCl (pH 7.5), 1%β-mercaptoethanol, 0.1% lauryl sarcosyl sodium salt). The disruptedsuspension was centrifuged at 10,000×G for 10 minutes to removeinsoluble matters. After that, a supernatant was overlaid on an equalamount of a density-equilibrating buffer (4M cesium chloride, 10 mMethylenediaminetetraacetic acid disodium salt pH 7.5) in aultracentrifugation tube, and it was centrifuged at 30,000 rpm at 20° C.for 18 hours to separate 600 μg of total RNA.

mRNA was prepared from the total RNA by using a POLY(A) QUICK mRNAextraction kit (produced by Stratagene) in accordance with a protocol ofthe kit. Namely, a part of the obtained total RNA (500 μg) was adsorbedto an oligo dT column. The column was washed twice with a higher saltbuffer (200 μl) and three times with a lower salt buffer (200 μl). Afterthat, an elution buffer (200 μl) was allowed to pass through the columnfour times at 65° C. to separate and purify mRNA (10 μg).

(2) Synthesis of cDNA

cDNA was synthesized by using a Time Savor DNA synthesis kit (producedby Pharmacia) in accordance with a protocol of the kit. Namely, thepurified mRNA (3 μg) was mixed with a first strand reaction solutioncontaining random hexamer primers (0.3 μg), 1 mM dithiothreitol, andreverse transcriptase, followed by a reaction at 37° C. for 1 hour tosynthesize a first strand.

The reaction solution was mixed with a second strand reaction solutioncontaining Escherichia coli RNase H and Escherichia coli DNA polymerase,followed by reactions at 12° C. for 30 minutes and at 22° C. for 1 hourto synthesize cDNA. Incubation was further performed at 65° C. for 10minutes. After that, the reaction solution was treated withphenol/chloroform to inactivate the enzyme activity. Next, a gelfiltration span column attached to the kit was used to performcentrifugation at 400×G for 2 minutes. Thus unreacted primers wereremoved to obtain double strand cDNA (3 μg).

(3) Preparation of cDNA Library

An EcoRI/NotI adapter attached to the Time Savor DNA kit was ligated toboth ends of the double strand cDNA obtained as described above inaccordance with a protocol of the kit. Namely, cDNA (3 μg), theEcoRI/NotI adapter (3 μl), a polyethylene glycol buffer (30 μl), an ATPsolution (1 μl), and T4 DNA ligase (1 μl) were mixed to perform aligation reaction at 16° C. for 1 hour. The reaction solution wasfurther incubated at 65° C. for 10 minutes to inactivate the enzymeactivity. After that, an ATP solution (1.5 μl) and T4 polynucleotidekinase (1 μl) were added, and reacted at 37° C. for 30 minutes tophosphatize the 5'-end of the adapter. After that, the reaction solutionwas incubated at 65° C. for 10 minutes, and treated withphenol/chloroform to inactivate the enzyme activity. Next, a gelfiltration span column attached to the kit was used to centrifuge thereaction solution at 400×G for 2 minutes. Thus unreacted adapter wasremoved.

cDNA with the ligated adapters at both ends was ligated with an EcoRIsite of a lambda phage vector λZAPII (produced by Stratagene) to preparerecombinant phage DNA. Namely, λZAPII/EcoRI/CIAP arm (1 μg) and aligation buffer (100 mM Tris-HCl (pH 7.6), 25 mM magnesium chloride, 300mM sodium chloride) were added to 400 ng of cDNA with the ligatedadapters, to which an enzyme solution (B solution, produced by TakaraShuzo, Ligation Kit) containing T4 DNA ligase was added in an equalamount to perform a ligation reaction at 26° C. for 10 minutes.

Recombinant phage DNA obtained as described above was packaged by usinga packaging kit GIGAPACKII GOLD (produced by Stratagene) in accordancewith a protocol of the kit. Namely, λZAPII arm DNA (3 μg) ligated withcDNA described above was mixed with a packaging extraction solution ofthe kit to execute packaging by performing a reaction at 22° C. for 2hours. This reaction solution was added with 500 μl of a phage dilutionsolution (0.58% sodium chloride, 0.2% magnesium sulfate, 50 mM Tris-HCl(pH 7.5), 0.01% gelatin).

The titer of the obtained recombinant phage was checked. After that, aphage library was prepared by using a half amount of the phage packagingreaction solution, and using Escherichia coli XL-1 Blue (produced byStratagene) as a recipient. Namely, 10 plates each having a diameter of150 mm containing a plaque formation medium (Bactotryptone 1%, yeastextract 0.5%, sodium chloride 0.5%, magnesium sulfate 1 mM, maltose0.2%) were prepared. The phage diluted with the phage dilution solution,and the recipient were plated on the 10 plates so that 20,000 plaqueswould be formed per one plate, followed by cultivation at 37° C. for 12hours to obtain the library of the recombinant phage.

<2> Preparation of Probe DNA for Isolating Objective Gene

(1) Amplification of Partial Fragment of Objective Gene by RT-PCR Method

The total RNA from Crotalus horridus horridus was used as a material toamplify DNA coding for the peptide capable of inhibiting the binding ofvon Willebrand factor with platelets in accordance with the RT-PCRmethod.

Based on the amino acid sequence of the peptide shown in SEQ ID NO: 2,its sections with less degeneracy of codons were selected to prepareprimers for RT-PCR (reverse transcription polymerase chain reaction).Primers were chemically synthesized by an entrusted company, Biologica.SEQ ID NOS: 4 and 5 show nucleotide sequences of these primers. However,in SEQ ID NO: 4, 3rd and 6th nucleotides are mixtures of A and G, and12th nucleotide is a mixture of T, C, A, and G. In SEQ ID NO: 5, 3rdnucleotide is a mixture of T, C, A, and G, 6th and 15th nucleotides aremixtures of T and C, and 9th nucleotide is a mixture of A and G.

RT-PCR was performed by using the primer described above for the totalRNA of Crotalus horridus horridus prepared in the same manner asdescribed above. In order to synthesize a first strand, the total RNA (5μg) was mixed with reverse transcriptase SUPERSCRIPT II (produced byGIBCO) (2.5 μl), a first strand buffer (20 μl) attached to the enzymesolution, 0.1M dithiothreitol (10 μl), and 10 mM dNTP (5 μl). A reactionwas performed at 42° C. for 1 hour to synthesize the first strand. Thereaction solution was incubated at 95° C. for 5 minutes to inactivatethe reverse transcriptase. Next, the first strand was used as a templateto perform the PCR process. Namely, the first strand reaction solution(5 μl), a PCR reaction buffer (10 μl), 10 mM dNTP (5 μl), the primers(each 800 pmol), Taq polymerase (10μ) were mixed to perform a reactionover 25 cycles by using DNA Thermal Cycler (produced by Perkin-Elmer),one cycle comprising periods at 95° C. for 0.5 minute, at 52° C. for 1minute, and at 72° C. for 2 minutes.

The PCR reaction solution was subjected to 2% agarose gelelectrophoresis to analyze amplified DNA. As a result, a band of DNA wasobserved at a position of about 300 base pairs.

(2) Determination of Nucleotide Sequence of Amplified Fragment

The DNA fragment amplified as described above was subcloned into aplasmid by using a pCR-ScriptSK(+) cloning kit (produced by Stratagene)in accordance with a protocol of the kit. Namely, the PCR reactionsolution was added and mixed with a ligation buffer, 1 mM ATP, pCRscript(produced by Stratagene) as a vector (10 ng), restriction enzyme SrfI (5units), and T4 DNA ligase to perform a ligation reaction at 25° C. for 1hour. After that, the reaction solution was incubated at 65° C. for 10minutes to inactivate ligase. This reaction product was used totransform E. coli DH5α by the competent cell method, followed by platingon an L-Ap plate (Bactotryptone 1%, yeast extract 0.5%, sodium chloride0.5%, sodium ampicillin 100 μg/ml) to perform cultivation at 37° C. for18 hours. Bacterial cells which formed colonies were separated from theplate, a part of which was cultivated in a liquid medium to prepare aplasmid in accordance with the alkaline method ("Molecular Cloning", 2ndedition, Vol. 1, published by Cold Spring Harbor Press). This plasmidwas designated as pCHAprobe.

The nucleotide sequence of the cloned fragment of pCHAprobe was analyzedby the dye Terminator method by using M13M4 or M13 reverse (produced byTakara Shuzo) as a primer, and using DNA Sequencer A373 (produced byApplied Biosystems) in accordance with the method of use of thesequencer. As a result, the cloned DNA fragment comprised 272 basepairs, and had a nucleotide sequence shown in SEQ ID NO: 6. When thesequence was translated into amino acids, the amino acids correspondedto those of a part of the objective peptide. Thus it was possible todemonstrate that the obtained cloned fragment was a part of theobjective gene of the peptide AS1051.

(3) Labeling of Probe

pCHAprobe was digested with restriction enzymes SacI and BamHI atcorresponding sites existing at both ends of the cloned insert fragment.A DNA fragment having a size of 340 base pairs was separated by 2%agarose gel electrophoresis. DNA was recovered by using a DNA recoverykit (Takara EASYTRAP, produced by Takara Shuzo) in accordance with aprotocol of the kit. This DNA (25 ng) was labeled with radioisotope byusing α-³² P!dCTP and a random primer labeling kit (produced by TakaraShuzo). Unreacted α-³² P!dCTP was removed from the labeling reactionsolution by using Nick column for gel filtration (produced by Pharmacia)to obtain a labeled probe.

<3> Preparation of Objective Gene by Plaque Hybridization

A gene coding for an entire length of the AS1051 peptide was screenedfrom the cDNA phage library in accordance with plaque hybridization byusing the probe described above.

Plaques of the λZAPII cDNA phage library were formed on a plate asdescribed above. The plaques were transferred from the plate to a nylonfilter Highbond-N (produced by Amersham) in accordance with a recipeattached to the filter. The filter is alkaline-treated to achieve lysisof phage. After that, phage DNA was immobilized to the filter by bakingat 80° C. for 2 hours.

The filter was hybridized with the ³² P-labeled probe (1×10⁶ cpm/ml) at37° C. for 16 hours in a solution containing 5×SSPE buffer (20×SSPC:3.6M sodium chloride, 0.2M sodium phosphate buffer pH 7.7, 20 mM EDTAdisodium salt), 30% formamide, 5×Denhardt's solution (100×Denhardt'ssolution: 2% bovine serum albumin, 2% Ficoll 400, 2% polyvinylpyrrolidone), and 0.5% SDS. After that, the filter was washed twice atroom temperature in 6×SSC (20×SSC: 3M sodium chloride, 0.3M trisodiumcitrate) and 0.1% SDS, and further it was washed twice at 50° C. in2×SSC and 0.1% SDS to remove the probe non-specifically bound with thefilter. An X-ray film HP20 (produced by Fuji Photo Film) was exposedwith the filter at -80° C. for 24 hours. Clones corresponding topositive spots on the film were isolated from the phage plate to providepositive clones in primary screening. Similar screening operation wasrepeated to obtain positive clones which formed single plaques.

The λZAPII cDNA phages of the obtained positive clones were infectedwith a helper phage ExAssist (produced by Stratagene). SOLR cells(produced by Stratagene) provided as non-amber suppressor Escherichiacoli were infected therewith. Thus strains of Escherichia coli wereobtained, which harbored plasmids containing the cDNA fragment insertedinto an EcoRI site of a plasmid pBluescriptSK(-) (produced byStratagene). The plasmids were prepared from cells of these strains inaccordance with the alkaline SDS method. The nucleotide sequence of theinserted fragment was determined by using DNA Sequencer A373 (producedby Applied Biosystems).

As a result, four positive clones had nucleotide sequences coding forthe objective peptide. The harbored plasmids were designated as pCHA1,pCHA2, pCHA3, and pCHA4 respectively. E. coli harboring pCHA1(HB101/pCHA, E. coli AJ13023) has been internationally deposited under adeposition number of FERM BP-4781 based on the Budapest Treaty sinceAug. 12, 1994 in National Institute of Bioscience and Human Technologyof Agency of Industrial Science and Technology of Ministry ofInternational Trade and Industry (postal code: 305, 1-3 Higashi-Icchome,Tsukuba-shi, Ibaraki-ken, Japan). SEQ ID NO: 7 in Sequence Listing showsa nucleotide sequence of the gene coding for the AS1051 peptide clonedas described above. SEQ ID NO: 8 shows an amino acid sequence of thepeptide encoded by the gene. This gene had a typical secretion signalcomprising 22 amino acids including methionine as an amino acid forinitiation of translation and following amino acids.

<4> Expression and Production of AS1051 Peptide by Using Escherichiacoli as Host and Preparation of Active Peptide

(1) Construction of Escherichia coli Expression Plasmid pCHAT7

The DNA fragment coding for the AS1051 peptide obtained as describedabove was introduced into a plasmid pGEMEX-1 (produced by Promega)containing T7 promoter to construct an expression plasmid forEscherichia coli (see FIG. 12).

At first, in order to facilitate the following operations, an NdeIcleavage site far from the T7 promoter was selected from two restrictionenzyme NdeI cleavage sites existing in pGEMEX-1, and it was delected toprepare a plasmid. Namely, pGEMEX-1 was partially digested with NdeI.Digested ends were blunt-ended by using DNA Blunting Kit (produced byTakara Shuzo). Circular plasmids were formed again by using Ligation Kit(produced by Takara Shuzo). Obtained plasmids were used to transformEscherichia coli. A plasmid having only one NdeI cleavage site existingat the objective position was obtained from transformants, which wasdesignated as pGEMEX(NdeI).

pCHA1 described above and pGEMEX(Nde1) were digested with restrictionenzymes NdeI and SacI to extract DNA fragments of 100 base pairs and 3.2kilo base pairs respectively by agarose gel electrophoresis. The DNAfragment of 100 base pairs originating from pCHA1 contained a 3'-endregion (corresponding to nucleotide numbers 490 to 559 in SEQ ID NO: 7)of the gene coding for the AS1051 peptide.

A ligation reaction for these DNA fragment was performed by usingLigation Kit (produced by Takara Shuzo). The reaction solution was usedto transform Escherichia coli HB101. A recombinant plasmid pCHA(NS) wasobtained from transformants separated on an ampicillin-containing plate.

Next, a 5'-end region (corresponding to nucleotide numbers 135 to 489 inSEQ ID NO: 7) of the gene coding for the AS1051 peptide was insertedinto the obtained plasmid pCHA(NS) to construct a plasmid for expressingand producing the AS1051 peptide in Escherichia coli.

In order to incorporate the 5'-end region of the gene coding for theAS1051 peptide into pCHA(NS), DNA primers were synthesized foramplifying the region in accordance with the PCR method. In thisprocedure, as for the primer for the 5'-end Bide, a primer containing anNdeI recognition sequence (CHANdeI primer: SEQ ID NO: 9) was used sothat the 5'-end of an amplified fragment had an NdeI site. This primeralso had a nucleotide sequence ATG as a translation initiation signal(nucleotide numbers 9 to 11 in SEQ ID NO: 9) before a codon of asparticacid as the N-terminal amino acid of the AS1051 peptide on the 5'-endside. It is noted that the initiation codon overlaps the NdeIrecognition sequence (nucleotide numbers 6 to 11 in SEQ ID NO: 9).

As for the primer for the 3'-end side, a primer containing a HindIIIrecognition sequence (CHAHindIII: SEQ ID NO: 10, the HindIII recognitionsequence corresponds to nucleotide numbers 10 to 15) was usedconsidering the construction of an expression plasmid to be used for anexpression system for cultured insect cells described below.

The gene coding for the AS1051 peptide was amplified in accordance withthe PCR process by using the primers described above. The PCR processwas repeated over 25 cycles, one cycle comprising periods at 94° C. for15 seconds, at 50° C. for 1 minute, and at 72 ° C. for 2 minutes. ThePCR reaction solution was treated with phenol/chloroform to inactivateTaq polymerase. The amplified DNA fragment comprising 400 base pairs waspurified in accordance with the ethanol precipitation method, and thenit was digested with the restriction enzyme NdeI. This DNA fragment wasligated with pCHA(NS) having been digested with the restriction enzymeNdeI by using Ligation Kit (produced by Takara Shuzo). An obtainedplasmid was used to transform E. coli HB101 strain in accordance withthe competent cell method. A transformant was cultivated for 16 hours onan ampicillin-containing plate.

A plasmid was prepared from the grown transformant in accordance withthe alkaline SDS method. The nucleotide sequence was determined by usingT7 primer and SP6 primer (produced by Stratagene) and using DNASequencer A373 (produced by Applied Biosystems). Thus it was confirmedthat the objective AS1051 peptide expression vector was constructed. Theconstructed expression vector was designated as pCHAT7. The plasmidconstruction process described above is shown in FIG. 12.

(2) Production of Peptide by Escherichia coli

In order to produce the AS1051 peptide by Escherichia coli by using theAS1051 peptide expression vector pCHAT7, E. coli JM109(DE3) (produced byPromega) was transformed with pCHAT7 in accordance with the competentcell method, which was cultivated at 25° C. for 2 days on anampicillin-containing plate to select a plasmid-harboring strain. It isnoted that E. coli JM109(DE3) is a strain having an RNA polymerase geneof T7 phage connected downstream from lacUV5 promoter, which isconstructed to efficiently express only T7 promoter such thattranscription by lacUV5 promoter is induced upon addition of IPTG(isopropyl-β-D-thiogalactopyranoside) to produce RNA polymerase of T7phage. Therefore, the plasmid-harboring strain efficiently expressesdthe AS1051 peptide.

E. coli JM109(DE3)/pCHAT7 harboring the expression plasmid wasinoculated to 10 Sakaguchi flasks each having a volume of 500 mlcontaining 100 ml of an LB-Ap medium (1% Bactotryptone, 0.5% yeastextract, 0.5% sodium chloride, 100 μg/ml ampicillin), and cultivated at30° C. for 16 hours with shaking. The inducing agent IPTG was added tothe medium to give a final concentration of 0.5 mM to further continuecultivation at 37° C. for 4 hours with shaking. After the cultivation,bacterial cells were collected by centrifugation, and then suspended in100 ml of a buffer (30 mM Tris-HCl pH 7.5, 10 mM EDTA(ethylenediaminetetraacetic acid disodium salt), 30 mM sodium chloride)to wash the bacterial cells. The cells were collected again bycentrifugation, and suspended in 20 ml of a cell disruption buffer (0.5MEDTA, pH 8). Egg white lysozyme (20 mg) was added to this suspension totreat it at 0° C. for 1 hour to disrupt cell walls of the cells. Thesuspension was further disrupted and treated with a ultrasonic disrupterInsonator 200M (produced by Kubota) at 180 W for 10 minutes. Aninsoluble fraction of the cells (inclusion bodies) was obtained bycentrifuging the disrupted suspension at 6,000 rpm for 20 minutes.

(3) Solubilization and Activation of Inclusion Bodies

Inclusion bodies obtained from 1 liter of culture liquid were dissolvedin 7M guanidine hydrochloride solution (28.6 ml) containing 10 mM EDTAand 0.5M Tris-HCl (pH 8.5), to which 71.4 ml of distilled water wasadded, followed by storage at 4° C. for 2 days to perform oxidation withair. After that, this solution was made acidic by adding 0.5 ml oftrifluoroacetic acid, and then insoluble matters were removed bycentrifugation. A supernatant was fractionated and collected byhigh-performance liquid chromatography (HPLC) by using a reverse phasecolumn (Vydac 214TP1022, produced by Vydac) to obtain recombinant AS1051(9.0 mg). The obtained purified peptide provided the same molecularweight as that of AS1051 described in Example 1 <4> onSDS-polyacrylamide gel electrophoresis. The inhibiting activity onplatelet aggregation approximately was measured for the obtainedrecombinant AS1051 by using the same method as described in Example1<5>. As a result, the ADP-induced aggregation and the collagen-inducedaggregation were not inhibited, while equivalent inhibiting activitieswere exhibited on the ristocetin-induced aggregation and thebotrocetin-induced aggregation as compared with AS1051 obtained inExample 1 <4>.

The amino acid sequence of the recombinant AS1051 obtained as describedabove was determined as follows. A solution (450 ml) of 2 mM EDTA and0.1M Tris-HCl (pH 8.5) containing 500 μg of the recombinant AS1051 wasadded with 1 μl of 4-vinylpyridine, and further added with 15 μg oflysylendopeptidase (produced by Wako Pure Chemical) to perform enzymaticdigestion at 37° C. for 3 hours. A digested product was subjected toreverse phase HPLC in the same manner as described in Example 1 <4> tofractionate digested fragment peptides. As a result of amino acidsequence analysis for each of the digested fragment peptides, it wasconfirmed that the recombinant AS1051 had an amino acid sequenceconstituted by the sequence of AS1051 shown in SEQ ID NO: 2 and amethionine residue bound to its amino terminal. As for the manner ofdisulfide bonds, the presence of a disulfide bond between Cys4 and Cys15was confirmed by mass spectrometry for a fragment peptide containing theboth residues. Further, the presence of disulfide bonds between Cys32and Cys120 and between Cys95 and Cys112 was confirmed by enzymaticallydigesting fragment peptides comprising the fragments A, B, C shown inFIG. 7 cross-linked through disulfide bonds by using V8 protease(produced by Wako Pure Chemical) (3 μg of V8 protease in a 0.1M ammoniumhydrogencarbonate solution), and analyzing amino acid sequences offragment peptides separated by HPLC using the reverse phase column inthe same manner as described in Example 1 <4>.

The decrease in platelets was not observed when the obtained recombinantAS1051 was intravenously administrated to mice in an amount of 1000μg/kg.

EXAMPLE 3 Production of Anti-Thrombus Single Strand Peptide byBaculovirus/Cultured Insect Cell Expression System

The anti-thrombus single strand peptide was produced by expressing theAS1051 gene obtained in Example 2 in a cultured insect cell expressionsystem.

<1> Construction of Baculovirus AS1051 Expression Vector

A baculovirus expression system was constructed by using a MaxBacbaculovirus expression system (produced by Invitrogen) (see FIG. 13).Namely, pCHA1 obtained in Example 2 was digested with a restrictionenzyme EcoRI, and its ends were blunt-ended by using DNA Blunting Kit(produced by Takara Shuzo) in accordance with a protocol of the kit.This fragment was further digested with a restriction enzyme PstI, anddigested products were subjected to agarose gel electrophoresis torecover a DNA fragment of 400 base pairs. On the other hand, anexpression vector pBlueBacIII was digested with a restriction enzymeHindIII, and its ends were blunt-ended by using DNA Blunting Kit. Thisfragment was further digested with a restriction enzyme PstI, anddigested products were subjected to agarose gel electrophoresis torecover a DNA fragment of 10.3 kilo base pairs. These recoveredfragments were ligated by using Ligation Kit, and E. coli HB101 wastransformed therewith in accordance with the competent cell method toselect a transformant on an ampicillin-containing plate. The constructedplasmid was designated as pCHAbac. The construction of the plasmid isshown in FIG. 13.

<2> Transformation of Cultured Insect Cells (Sf9 Strain) with pCHAbacand Preparation of Recombinant Virus

Cultured insect cells were transformed and a recombinant virus wasobtained in accordance with a protocol of the MaxBac baculovirusexpression system (produced by Invitrogen). Namely, DNA (1 μg) of a wildtype nuclear polyhedrosis virus (Autographa californica nuclearpolyhedrosis virus: AcMNPV) and pCHAbac (3 μg) were introduced intocultured insect cells (Spodoptera frugiperda 9 strain (Sf9 strain)) inaccordance with the liposome method. The virus-introduced cells werecultivated in a TNM--FH (FBS+) culture liquid (produced by Invitrogen)at 27° C. for 48 hours. After that, the culture liquid was recovered toobtain a virus solution.

Cultured insect cells (Sf9 strain) were infected with the virussolution, and cultivated at 27° C. for 7 days on an FNM--FH (FBS+) softagar medium containing 150 μg/ml of X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside). Cells (blue color), whichwere infected with a virus undergone recombination between the wild typeAcMNPV and pCHAbac, were selected. The cells were picked up from theplate by using a Pasteur pipet, and Sf9 cells were infected therewithagain after appropriate dilution. The purifying operation as describedabove was repeated twice to obtain a virus solution in which only therecombinant virus was present.

<3> Expression of AS1051 Peptide by Baculovirus/cultured Insect CellExpression System

The AS1051 peptide was expressed by using the recombinant virus. Sf9cells (6×10⁶ cells) were inoculated to 10 ml of an FNM--FH (FBS+)culture liquid in a flask for cell culture (NUNCLON 260 ml, bottom area:75 cm², produced by Nunc), and the purified recombinant virus (3×10⁶cfu) was added thereto to perform cultivation at 27° C. for 24 hours.After that, the culture liquid was removed from the flask to which 10 mlof an FNM--FH (FBS--) culture liquid containing no fetal bovine serumwas added to perform further cultivation at 27° C. for 7 days. Aftercompletion of the cultivation, 4 ml of the culture liquid was collected,which was concentrated into 400 μl by using Centricon 10 (produced byAmicon) to obtain a culture supernatant concentrate (10-foldconcentrated solution).

On the other hand, a buffer (30 mM Tris-HCl pH 7.5, 10 mM EDTA, 30 mMsodium chloride) was added to the flask from which the culture liquidhad been removed. Cells were exfoliated from flask walls by pipetting.After that, a cell suspension was recovered, and centrifuged to wash thecells once. The cells were subsequently resuspended in 2 ml of thebuffer. This cell suspension was treated with a ultrasonic disrupterInsonator 200M (produced by Kubota) at 180 W for 5 minutes to disruptthe cells. After the disruption, centrifugation was performed at10,000×G for 30 minutes, and a supernatant was recovered to obtain acell-disrupted solution. Control sample were also provided bycultivating Sf9 cells infected with the wild type AcNMPV virus and Sf9cells infected with no virus under the same condition as described abovefor sample preparation.

The culture supernatant concentrate (5 μl) and the cell-disruptedsolution (10 μl) prepared as described above were subjected toSDS-polyacrylamide gel electrophoresis respectively under a reducedcondition with addition of 1% mercaptoethanol, followed by staining withCoomassie Brilliant Blue (CBB). As a result, a protein was detected at aposition of 15 kilodaltons in considerable amounts in both of theculture supernatant concentrate and the cell-disrupted solution obtainedfrom the cells infected with the recombinant virus. On the contrary, noprotein could be detected at the position of 15 kilodaltons in both ofthe controls including the culture supernatant concentrate and thecell-disrupted solution obtained from the Sf9 cells only, and thoseobtained from the Sf9 cells infected with the wild type virus AcMNPV. Itis noted that the value of the molecular weight of 15 kilodaltonscoincides with the molecular weight of AS1051 purified from the crudesnake venom.

<4> Activity of AS1051 Peptide Produced by Baculovirus/Cultured InsectCell Expression System

The recombinant protein in cultured insect cells and the recombinantprotein in culture supernatant obtained as described above provided theinhibition on the binding of von Willebrand factor with plateletsinduced by botrocetin, as measured by the following method.

Formalin-fixed platelets were prepared as follows. Fresh blood collectedfrom healthy human added with 1/10 volume of 3.8% sodium citrate wascentrifuged at 900 rpm for 15 hours to obtain human platelet rich plasma(PRP) to which a 0.15M sodium chloride aqueous solution having the samevolume and containing 20 mM phosphate buffer (pH 7.4) dissolved with 2%paraformaldehyde was added, followed by being stored at 4° C. overnightstationarily and. After the storage, platelets were recovered bycentrifugation, and they were washed twice with a 0.15M sodium chlorideaqueous solution containing 20 mM phosphate buffer (pH 7.4). After thewashing, the fixed platelets were suspended in the same solution, andstored.

A test to measure the inhibition on the binding of ¹²⁵ I-labeled vonWillebrand factor with fixed platelets was performed by using the fixedplatelets obtained as described above in accordance with the method ofChopek et al. (M. W. Chopek et al., Biochemistry, 25, 3146-3155 (1986)).Namely, a suspension of the prepared fixed platelets was added with asample for measurement, botrocetin, and ¹²⁵ I-labeled von Willebrandfactor, and reacted at room temperature for 30 minutes to measure theamount of von Willebrand factor bound with the platelets by using aγ-counter (Packard Multi-Prias, produced by Packard). In thismeasurement, the reaction solution had a volume of 50 μl including 5 μlof the added sample for measurement.

FIG. 14 shows a result of measurement of the inhibiting activity on thebinding of von Willebrand factor with the platelets induced bybotrocetin with respect to cell-disrupted solutions prepared from theSf9 cells infected with the AS1051 recombinant virus and the control Sf9cells in accordance with the method described above. FIG. 15 shows aresult of measurement of the inhibiting activity on the binding of vonWillebrand factor with the platelets induced by botrocetin with respectto culture supernatant concentrates (3-fold concentrated solutions)prepared from the Sf9 cells infected with the AS1051 recombinant virusand the control Sf9 cells in accordance with the method described above.As clarified from these results, the cell-disrupted solution and thecultured cell supernatant obtained from the cells infected with therecombinant virus almost completely inhibited the binding of vonWillebrand factor to the platelets. According to this fact, it wasrevealed that the AS1051 peptide to be produced by thebaculovirus/cultured insect cell expression system as an intracellularsoluble protein and an extracellular secretion protein has the activityto inhibit the binding between platelets and von Willebrand factor.

EXAMPLE 4 Production of Anti-Thrombus Single Strand Peptide by CulturedAnimal Cell Expression System

The anti-thrombus single strand peptide was produced by expressing theAS1051 gene in a cultured animal cell expression system by using CHOcells.

The AS1051 gene was excised by digesting pCHA1 with a restriction enzymePstI. Both ends of an obtained DNA fragment were blunt-ended with DNABlunting Kit (produced by Takara Shuzo). An XhoI linker (produced byTakara Shuzo) was ligated to the both ends. This DNA fragment wasdigested with a restriction enzyme XhoI, and then subjected to agarosegel electrophoresis to extract a DNA fragment of 500 base pairs. The DNAfragment was inserted into an XhoI site of a CHO cell expression vectorpSD(X) (M. Murata et al., Proc. Natl. Acad. Sci. U.S.A., 85, 2434-2438(1988)). It is noted that this vector pSD(X) can express a foreign genein CHO cells, the vector comprising replication origins of pBR322 andSV40, an ampicillin resistance gene, and a methotrexate resistance gene,and the vector further comprising an SV40 promoter, an SV40 splicingsignal, and a poly(A) addition signal. The construction process of theplasmid described above is shown in FIG. 16.

Escherichia coli HB101 was transformed with pSD(X) harboring theinserted AS1051 gene to obtain a transformant. An expression plasmid, inwhich the AS1051 gene was inserted in a desired direction with respectto the vector, was obtained from the transformant. The plasmid wasdesignated as pCHASDX. The plasmid pCHASDX was used to transform adihydrofolate reductase-deficient strain of CHO cells in accordance withthe calcium phosphate method ("Current Protocols in Molecular Biology",Green Publishing Associates). Transformants were cultivated in a mediumcontaining alpha-MEM (nucleic acid minus) (produced by GIBCO), 10% fetalbovine serum (FCS), and methotrexate 0.05 μM. Thus transformantsharboring the expression plasmid incorporated in chromosome wereselectively grown.

Obtained strains were cultivated by increasing the concentration ofmethotrexate in the medium stepwise up to 0.5 μM to selectmethotrexate-resistant strains. Thus a strain was obtained in which theAS1051 gene was considered to be amplified on chromosome of CHO cells. Asingle clone of this strain was proliferated to obtain cells. Total RNAwas extracted from the cells by using ISOGEN kit (produced by NipponGene). The AS1051 gene was amplified by using primers CHA16Y (SEQ ID NO:13) and CHA115Q (SEQ ID NO: 14) in accordance with the RT-PCR method inthe same manner as described in Example 2 <2>. Amplified DNA wasanalyzed by agarose gel electrophoresis. As a result, it was detectedthat a DNA fragment having a size postulated from the nucleotidesequence of the AS1051 gene was amplified. Thus it was confirmed thatmRNA of the AS1051 gene was transcribed in CHO cells. The cells wereused to investigate expression of the AS1051 peptide. Namely, the cells(2×104 cells) were cultivated for 4 days in 5 ml of a medium containingalpha-MEM (nucleic acid minus), 10% fetal bovine serum, and methotrexate0.5 μM. After that, the medium was exchanged with a serum-free mediumAS104 (produced by Ajinomoto) containing methotrexate 0.5 μM, followedby cultivation for further 3 days.

The medium was recovered after completion of the cultivation, and 4 mlof the medium was concentrated into 100 μl by using Centricon-10(produced by Amicon) to obtain a concentrate. The concentrate wassuccessively diluted to measure the activity to inhibit the binding ofvon Willebrand factor with platelets evoked by ristocetin and botrocetinin accordance with the method described in Example 3<4>. FIGS. 17 and 18show the inhibiting activities of solutions obtained by concentrating ordiluting, into predetermined concentrations, the culture supernatants ofAS1051 peptide-producing cells and control cells which did not producethe AS1051 peptide. As shown in FIGS. 17 and 18, it was demonstratedthat the inhibiting activity of the culture supernatant of thepeptide-producing cells depended on the concentration, and the activeAS1051 was contained.

EXAMPLE 5 Production of Mutant AS1051 Peptide

Escherichia coli was allowed to express an AS1051 peptide havingmutation (hereinafter referred to as "Cys81Ala mutation") to substitutean alanine residue for an 81th cysteine residue as counted from theN-terminal (except for the methionine residue to initiate translation)in order to investigate the activity to inhibit the binding of vonWillebrand factor with platelets.

<1> Production of Cys81Ala Mutant Peptide

Mutation was introduced into the AS1051 gene so that the cysteineresidue, which did not participate in disulfide bond formation of theAS1051 peptide (81th cysteine residue in SEQ ID NO: 2), was substitutedwith alanine in accordance with the site-directed mutagenesis fornucleotide sequence described in "PCR Protocol" (published by AcademicPress). pCHA1 was used as a template to perform the PCR process by usingthe primer CHANdeI (SEQ ID NO: 9) synthesized in Example 2 and a newlysynthesized primer CHAAlaF (SEQ ID NO: 11), or by using a newlysynthesized primer CHAAlaR (SEQ ID NO: 12) and the primer CHAHindIII(SEQ ID NO: 10) synthesized in Example 2. Respective reaction productswere subjected to agarose gel electrophoresis, and amplified DNAfragments were extracted from the gel. These DNA fragments were used astemplates to perform the second PCR process by using the primers CHANdeIand CHAHindIII to prepare a mutant gene. PCR-amplified DNA fragmentswere digested with a restriction enzyme NdeI, followed by agarose gelelectrophoresis to extract a DNA fragment of 360 bp from the gel. ThisDNA fragment was inserted into an NdeI site of PCHA(NS) having beendigested with the restriction enzyme NdeI. The plasmid constructionprocess described above is shown in FIG. 19.

The plasmid prepared as described above was used to transformEscherichia coli HB101 in accordance with the competent cell method.Transformants were selected on an ampicillin-containing plate. Plasmidswere prepared from the transformants in accordance with the alkaline SDSmethod. Nucleotide sequences were determined in accordance with themethod described in Example 8 by using T7 primer and SP6 primer(produced by Stratagene). Thus a plasmid, in which the objectivemutation was introduced, was selected. The obtained expression vectorwas designated as pCHA7Ala. pCHA7Ala was used to transform Escherichiacoli JM109(DE3) to obtain a transformant. The AS1051 peptide having theCys81Ala mutation was expressed and produced by cultivating thetransformant in the same manner as in Example 2<4>. Bacterial proteinswere subjected to SDS-polyacrylamide gel electrophoresis. As a result,it was confirmed that the recombinant peptide could be produced andaccumulated as inclusion bodies in cells of Escherichia coli inapproximately the same amount as that of the genetically recombined wildtype AS1051 peptide produced in Example 2 <4>.

<2> Solubilization and Activation of Inclusion Bodies of Cys81Ala MutantAS1051 Peptide

The inclusion bodies were solubilized and activated in accordance withthe method described in Example 2<4>. An obtained purified proteinexhibited the same molecular weight as the molecular weight of AS1051shown in Example 1<4> according to SDS electrophoresis. It was alsoconfirmed that the purified protein had the same manner of disulfidebonds as that of AS1051. The activity to inhibit platelet aggregationwas measured for the obtained Cys81Ala mutant peptide in accordance withthe same method as described in Example 1<5>. As a result, the mutantpeptide did not inhibit the ADP-induced aggregation and thecollagen-induced aggregation, and it inhibited the ristocetin-inducedaggregation and the botrocetin-induced aggregation at approximately thesame degree as that of AS1051. The mutant peptide exhibited theinhibiting activity equivalent to those of AS1051 obtained in Example1<4> and recombinant AS1051 obtained in Example 2<4> on the binding ofvon Willebrand factor with the fixed platelets induced by ristocetin orbotrocetin (FIG. 20). The mutant AS1051 peptide thus obtained was stablein storage in a solution at 30° C. and dialysis at 4° C., while therecombinant AS1051 peptide was somewhat insolubilized in storage in asolution at 30° C. on dialysis at 4° C. According to this result, it isconsidered that the recombinant mutant AS1051 peptide has higherstability than the recombinant AS1051 peptide.

EXAMPLE 6 Production of Shortened AS1051 Peptides

AS1051 peptides having shortened N-terminal portions and/or a shortenedC-terminal portion were expressed in Escherichia coli to investigate theactivities of the shortened AS1051 peptides to inhibit the binding ofvon Willebrand factor with platelets.

<1> Production of Shortened AS1051 Peptides

Expression systems for peptides were constructed starting from theAS1051 peptide having the Cys81Ala mutation (hereinafter referred to as"AS1051Cys81Ala peptide"), the constructed peptides being deficient in15 amino acid residues or 65 amino acid residues having been located onan N-terminal region and/or 11 amino acid residues having been locatedon a C-terminal region. Namely, the produced peptides were five speciesincluding a peptide (AS1051A-1) having a sequence from a 16th tyrosineresidue to a 126th arginine residue, a peptide (AS1051A-2) having asequence from a 67th tyrosine residue to the 126th arginine residue, apeptide (AS1051A-3) having a sequence from a 1st aspartic acid residueto a 115th glutamine residue, a peptide (AS1O51A-4) having a sequencefrom the 16th tyrosine residue to the 115th glutamine residue, and apeptide (AS1051A-5) having a sequence from the 67th tyrosine residue tothe 115th glutamine residue. Structures of these peptides areschematically shown in FIG. 21. The numbers of amino acids were countedwhile excluding methionine for initiation of translation, the numbersindicating amino acid numbers in the amino acid sequence shown in SEQ IDNO: 2.

Expression plasmids for the shortened AS1051 peptides were constructedas follows. pCHAT7Ala was used as a template to perform the PCR processby using primers CHA16Y (SEQ ID NO: 13) and CHAHindIII (SEQ ID NO: 10)for AS1051A-1, using primers CHA67Y (SEQ ID NO: 15) and CHAHindIII forAS1051A-2, using primers CHANdeI (SEQ ID NO: 9) and CHA115Q (SEQ ID NO:14) for AS1O51A-3, using primers CHA16Y and CHA115Q for AS1051A-4, andusing primers CHA67Y and CHA115Q for AS1051A-5.

As for amplified products obtained by using the primers CHA16Y andCHAHindIII and the primers CHA67Y and CHAHindIII, the respectiveamplified products were digested with NdeI, and subjected to agarose gelelectrophoresis to extract DNA fragments of 310 base pairs and 160 basepairs respectively. The respective DNA fragments were inserted into anNdeI site of pCHA(NS). Obtained recombinant plasmids were used totransform E. coli HB101 in accordance with the calcium chloride method,and transformants were selected on ampicillin-containing plates.Objective transformants, in which the respective amplified DNA fragmentswere inserted in objective directions with respect to the plasmids, wereselected from the transformants. An expression plasmid thus obtainedhaving the inserted fragment of 310 base pairs was designated aspCHAT7Ala(16Y126R), and an expression plasmid thus obtained having theinserted fragment of 160 base pairs was designated aspCHAT7Ala(67Y126R).

As for amplified products obtained by using the primers CHANdeI andCHA115Q, the primers CHA16Y and CHA115Q, and the primers CHA67Y andCHA115Q, the respective amplified products were digested with NdeI andHindIII, and subjected to agarose gel electrophoresis to extract DNAfragments of 360 base pairs, 310 base pairs, and 160 base pairsrespectively. The respective DNA fragments were ligated with pCHA(NS)having been digested with NdeI and HindIII. Obtained recombinantplasmids were used to transform E. coli HB101 in accordance with thecalcium chloride method, and transformants were selected onampicillin-containing plates. An expression plasmid thus obtained havingthe inserted fragment of 360 base pairs was designated aspCHAT7Ala(1D115Q), an expression plasmid thus obtained having theinserted fragment of 310 base pairs was designated aspCHAT7Ala(16Y115Q), and an expression plasmid thus obtained having theinserted fragment of 160 base pairs was designated aspCHAT7Ala(67Y115Q).

The five species of the expression plasmids for the shortened AS 1051peptides were used to transform E. coli JM09(DE3) to obtaintransformants respectively. These transformants were cultivated in thesame manner as the method described in Example 2, and bacterial cellsafter the cultivation were microscopically observed. As a result, it wasconfirmed that all of the five species of the transformants formedinclusion bodies. Bacterial proteins of the respective transformantswere analyzed by SDS-polyacrylamide gel electrophoresis. As a result,considerable amounts of proteins were found at positions of molecularweights postulated from the respective amino acid sequences of theshortened AS10151 peptides.

The inclusion bodies were prepared from the respective transformants inthe same manner as described in Example 2. The inclusion bodies weredissolved in 286 μl of a 7M guanidine hydrochloride solution containing10 mM EDTA and 0.5M Tris-HCl (pH 8.5), and then stored at 4° C.overnight. After that, each of aliquots (10 μl) was applied tohigh-performance liquid chromatography by using an SSC-VP318-1251 column(diameter: 4.6 mm, length: 250 mm, produced by Senshu Kagaku). Elutionwas performed by using a concentration gradient from an acetonitrileconcentration of 31% to a concentration of 52% containing 0.1%trifluoroacetic acid. As a result, peaks originating from the inclusionbodies were obtained as shown in FIG. 22 (AS1051A-1), FIG. 23(AS1051A-2), FIG. 24 (AS1051A-3), FIG. 25 (AS1051A-4), and FIG. 26(AS1051A-5) respectively.

Remaining entire amounts of AS1051A-1, AS1O51A-2, AS1051A-3, AS1051A-4,and AS1051A-5 dissolved in the 7M guanidine hydrochloride solution andstored overnight as described above were made to have acidic pH adjustedwith trifluoroacetic acid. After that, they were fractionated andcollected by HPLC with a reverse phase column (Vydac 214TP1022, producedby Vydac, diameter: 22 mm, length: 250 mm) at a flow rate of 15 ml inaccordance elution with a concentration gradient (20 minutes) from anacetonitrile concentration of 30% to a concentration of 60% containing0.1% trifluoroacetic acid. Obtained respective peptides were added withbovine serum albumin in 10-fold amounts of the peptide, lyophilized, anddissolved in a physiological saline solution. These solutions were usedas samples to measure the activity to inhibit the binding of vonWillebrand factor with formalin-fixed platelets induced by botrocetin orristocetin in the same manner as the method described in Example 1<5>.Results are shown in FIG. 27. As shown in FIG. 27, all of the shortenedAS1051 peptides clearly exhibited the binding-inhibiting activity atconcentrations shown in FIG. 27.

Industrial Applicability

The present invention provides the peptide which inhibits the binding ofvon Willebrand factor with platelets without causing the decrease inplatelets, although the peptide is obtained from a peptide originatingfrom a snake venom which causes the decrease in platelets upon in vivoadministration, provided that the binding closely participates in crisisof thrombosis. Accordingly, it is possible to provide a pharmaceuticalcomposition which is hopeful as an anti-thrombosis drug.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 15    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AspLeuGluCysProSerGlyTrpSerSerTyrAspArgTyrCysTyr    151015    LysProPheLysGlnGluMetThrTrpAlaAspAlaGluArgPheCys    202530    SerGluGlnAlaLysGly    35    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 126 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    AspLeuGluCysProSerGlyTrpSerSerTyrAspArgTyrCysTyr    151015    LysProPheLysGlnGluMetThrTrpAlaAspAlaGluArgPheCys    202530    SerGluGlnAlaLysGlyGlyHisLeuLeuSerValGluThrAlaLeu    354045    GluAlaSerPheValAspAsnValLeuTyrAlaAsnLysGluTyrLeu    505560    ThrArgTyrIleTrpIleGlyLeuArgValGlnAsnLysGlyGlnPro    65707580    CysSerSerIleSerTyrGluAsnLeuValAspProPheGluCysPhe    859095    MetValSerArgAspThrArgLeuArgGluTrpPheLysValAspCys    100105110    GluGlnGlnHisSerPheIleCysLysPheThrArgProArg    115120125    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    AspCysProSerAspTrpSerSerTyrGluGlyHisCysTyrArgVal    151015    PheGlnGlnGluMet    20    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other..synthetic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CARGARATGACNTGGGC17    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other..synthetic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    TCNACYTTRAACCAYTC17    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 272 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Crotalus horridus horridus    (B) STRAIN:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    CAGGAGATGACTTGGGCCGATGCAGAGAGGTTCTGCTCGGAGCAGGCGAAGGGCGGGCAT60    CTCCTCTCTGTCGAAACCGCCCTAGAAGCATCCTTTGTGGACAATGTGCTCTATGCGAAC120    AAAGAGTACCTCACACGTTATATCTGGATTGGACTGAGGGTTCAAAACAAAGGACAGCCA180    TGCTCCAGCATCAGTTATGAGAACCTGGTTGACCCATTTGAATGTTTTATGGTGAGCAGA240    GACACAAGGCTTCGTGAGTGGTTCAAAGTCGA272    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 690 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Crotalus horridus horridus    (B) STRAIN:    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 66..512    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    CTGAGCAGACTTGCTACCTGTGGAGGCCGAGGAACAGTTCTCTCTGCAGGGAAGGAAAGA60    ACGCCATGGGGCGATTCATCTTCGTGAGCTTCAACTTGCTGGTCGTGTTC110    MetGlyArgPheIlePheValSerPheAsnLeuLeuValValPhe    151015    CTCTCCCTAAGTGGAACTCTAGCTGATTTGGAATGTCCCTCCGGTTGG158    LeuSerLeuSerGlyThrLeuAlaAspLeuGluCysProSerGlyTrp    202530    TCTTCCTATGATCGGTATTGCTACAAGCCCTTCAAACAAGAGATGACC206    SerSerTyrAspArgTyrCysTyrLysProPheLysGlnGluMetThr    354045    TGGGCCGATGCAGAGAGGTTCTGCTCGGAGCAGGCGAAGGGCGGGCAT254    TrpAlaAspAlaGluArgPheCysSerGluGlnAlaLysGlyGlyHis    505560    CTCCTCTCTGTCGAAACCGCCCTAGAAGCATCCTTTGTGGACAATGTG302    LeuLeuSerValGluThrAlaLeuGluAlaSerPheValAspAsnVal    657075    CTCTATGCGAACAAAGAGTACCTCACACGTTATATCTGGATTGGACTG350    LeuTyrAlaAsnLysGluTyrLeuThrArgTyrIleTrpIleGlyLeu    80859095    AGGGTTCAAAACAAAGGACAGCCATGCTCCAGCATCAGTTATGAGAAC398    ArgValGlnAsnLysGlyGlnProCysSerSerIleSerTyrGluAsn    100105110    CTGGTTGACCCATTTGAATGTTTTATGGTGAGCAGAGACACAAGGCTT446    LeuValAspProPheGluCysPheMetValSerArgAspThrArgLeu    115120125    CGTGAGTGGTTTAAAGTTGACTGTGAACAACAACATTCTTTCATATGC494    ArgGluTrpPheLysValAspCysGluGlnGlnHisSerPheIleCys    130135140    AAGTTCACGCGACCACGTTAAGATCCGGCTGTGTGAAGTCTGGAGAAG542    LysPheThrArgProArg    145    CAAGGAAGCCCCCCACCTCTCCCCACCCCCCACCTTCCGCAATCTCTGCTCTTCCCCCTT602    TGCTCAGTGGATGCTCTCTGTAGCCGGATCTGGGTTTTCTGCTCCAGATGGGTCAGAAGA662    TCCAATAAATTCTGCCTACCCAAAAAAA690    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 149 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    MetGlyArgPheIlePheValSerPheAsnLeuLeuValValPheLeu    151015    SerLeuSerGlyThrLeuAlaAspLeuGluCysProSerGlyTrpSer    202530    SerTyrAspArgTyrCysTyrLysProPheLysGlnGluMetThrTrp    354045    AlaAspAlaGluArgPheCysSerGluGlnAlaLysGlyGlyHisLeu    505560    LeuSerValGluThrAlaLeuGluAlaSerPheValAspAsnValLeu    65707580    TyrAlaAsnLysGluTyrLeuThrArgTyrIleTrpIleGlyLeuArg    859095    ValGlnAsnLysGlyGlnProCysSerSerIleSerTyrGluAsnLeu    100105110    ValAspProPheGluCysPheMetValSerArgAspThrArgLeuArg    115120125    GluTrpPheLysValAspCysGluGlnGlnHisSerPheIleCysLys    130135140    PheThrArgProArg    145    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other..synthetic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GGCGGCATATGGATTTGGAATGTCCCTCCGGTTG34    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other..synthetic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    TATCTCGAGAAGCTTACAGCCGGATCTTAACGTGGTCGCG40    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other..synthetic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    GATGCTGGAGGCTGGCTGTCCTTTGT26    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other..synthetic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    GGACAGCCAGCCTCCAGCATCAGTTA26    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 35 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other..synthetic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    GGTGGATCCATATGTACAAGCCCTTCAAACAAGAG35    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other..synthetic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    CTCAGAAAGCTTTTATTGTTGTTCACAGTCAACTTT36    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 35 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other..synthetic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GGTGGATCCATATGTATATCTGGATTGGACTGAGG35    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified monomeric polypeptidecomprising:(1) the amino acid sequence from residue 67 to residue 115 ofSEQ ID NO: 2, or (2) the amino acid sequence from residue 67 to residue115 of SEQ ID NO: 2 in which the cysteine residue at position 81 of SEQID NO: 2 is substituted with an amino acid other than cysteine, whereinthe polypeptide has at least one disulfide bond between two cysteineresidues in the amino acid sequence.
 2. The polypeptide of claim 1,which has a disulfide bond between the cysteine residues at positions 95and 112 of SEQ ID NO:
 2. 3. The polypeptide of claim 2, comprising theamino acid sequence from residue 67 to residue 115 of SEQ ID NO: 2 inwhich the cysteine residue at position 81 of SEQ ID NO: 2 is substitutedwith an amino acid other than cysteine.
 4. The polypeptide of claim 3,wherein the amino acid other than cysteine is alanine.
 5. Thepolypeptide of claim 1, comprising the amino acid sequence from residue16 to residue 1I5 of SEQ ID NO:
 2. 6. The polypeptide of claim 5, whichhas a disulfide bond between the cysteine residues at positions 95 and112 of SEQ ID NO:
 2. 7. The polypeptide of claim 5, which has disulfidebonds between the cysteine residues at positions 95 and 112 andpositions 32 and 120 of SEQ ID NO:
 2. 8. The polypeptide of claim 1,comprising the amino acid sequence from residue 1 to residue 115 of SEQID NO:
 2. 9. The polypeptide of claim 8, which has disulfide bondsbetween the cysteine residues at positions 95 and 112 and positions 4and 15 of SEQ ID NO:
 2. 10. The polypeptide of claim 1, comprising theamino acid sequence from residue 67 to residue 126 of SEQ ID NO:
 2. 11.The polypeptide of claim 10, which has a disulfide bond between thecysteine residues at positions 95 and 112 of SEQ ID NO:
 2. 12. Thepolypeptide of claim 10, which has disulfide bonds between the cysteineresidues at positions 4 and 15, positions 95 and 112, and positions 32and 120 of SEQ ID NO:
 2. 13. The polypeptide of claim 1, comprising theamino acid sequence from residue 16 to residue 126 of SEQ ID NO:
 2. 14.The polypeptide of claim 1, comprising the amino acid sequence fromresidue 1 to residue 126 of SEQ ID NO:
 2. 15. The polypeptide of claim1, which inhibits the binding between von Willebrand factor andplatelets.
 16. The polypeptide of claim 15, which does not cause asubstantial decrease in the number of platelets at a minimum dose forinhibiting the binding between von Willebrand factor and platelets whenthe polypeptide is administered in vivo.
 17. An isolated and purifiedDNA fragment encoding the polypeptide of claim
 1. 18. A recombinantvector comprising the DNA fragment of claim
 17. 19. A host celltransformed with the recombinant vector of claim
 18. 20. The transformedhost cell of claim 19, which is Escherichia coli, a cultured insect cellor a cultured animal cell.
 21. A method of producing the polypeptide ofclaim 1, comprising culturing the transformed host cell of claim 19 in asuitable culture medium to produce the polypeptide, followed byisolating the polypeptide from the culture medium.
 22. A method ofproducing the monomeric polypeptide of claim 1 from a dimericpolypeptide which is obtainable from the venom of Crotalus horridushorridus, wherein the dimeric polypeptide has two polypeptide chainsthat are linked by at least one inter-chain disulfide bond, said methodcomprising:exposing the multimeric polypeptide to a protein-denaturingagent and a reducing agent selected from the group consisting ofglutathione and cysteine, thereby reducing the inter-chain disulfidebond to produce the monomeric polypeptide of claim 1, followed byisolating the monomeric polypeptide.
 23. A method of producing themonomeric polypeptide of claim 1, comprising:culturing Escherichia colitransformed with the vector of claim 1 in a suitable culture medium toaccumulate the polypeptide in the Escherichia coli, exposing thepolypeptide accumulated in the Escherichia coli to a protein-denaturingagent, followed by generating intra-molecular disulfide bonds within thechains of the polypeptide by removing the protein denaturing agent or bydecreasing the concentration of the protein denaturing agent.
 24. Apharmaceutical composition comprising a therapeutically effective amountof the polypeptide of claim 1 or a pharmaceutically acceptable salt ofthe polypeptide.
 25. A method of inhibiting the binding between bindingbetween von Willebrand factor and platelets in a patient in needthereof, comprising administering to the patient an effective amount ofthe polypeptide of claim
 1. 26. A method of producing an anti-thrombuseffect in a patient in need thereof, comprising administering to thepatient an effective amount of the polypeptide of claim 1.